CRISPR/Cas9 genome-editing applied to MdPGT1 in apple results in reduced foliar phloridzin without impacting plant growth.

Phloridzin is the most abundant polyphenolic compound in apple (Malus × domestica Borkh.), which results from the action of a key phloretin-specific UDP-2'-O-glucosyltransferase (MdPGT1). Here, we simultaneously assessed the effects of targeting MdPGT1 by conventional transgenesis and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing. To this end, we conducted transcriptomic and metabolic analyses of MdPGT1 RNA interference knockdown and genome-edited lines. Knockdown lines exhibited characteristic impairment of plant growth and leaf morphology, whereas genome-edited lines exhibited normal growth despite reduced foliar phloridzin. RNA-sequencing analysis identified a common core of regulated genes, involved in phenylpropanoid and flavonoid pathways. However, we identified genes and processes differentially modulated in stunted and genome-edited lines, including key transcription factors and genes involved in phytohormone signalling. Therefore, we conducted a phytohormone profiling to obtain insight into their role in the phenotypes observed. We found that salicylic and jasmonic acid were increased in dwarf lines, whereas auxin and ABA showed no correlation with the growth phenotype. Furthermore, bioactive brassinosteroids were commonly up-regulated, whereas gibberellin GA4 was distinctively altered, showing a sharp decrease in RNA interference knockdown lines. Expression analysis by reverse transcriptase-quantitative polymerase chain reaction expression analysis further confirmed transcriptional regulation of key factors involved in brassinosteroid and gibberellin interaction. These findings suggest that a differential modulation of phytohormones may be involved in the contrasting effects on growth following phloridzin reduction. The present study also illustrates how CRISPR/Cas9 genome editing can be applied to dissect the contribution of genes involved in phloridzin biosynthesis in apple.

Distinct and Temporally Stable Assembly Mechanisms Shape Bacterial and Fungal Communities in Vineyard Soils.

Microbial communities in agricultural soils are fundamental for plant growth and in vineyard ecosystems contribute to defining regional wine quality. Managing soil microbes towards beneficial outcomes requires knowledge of how community assembly processes vary across taxonomic groups, spatial scales, and through time. However, our understanding of microbial assembly remains limited. To quantify the contributions of stochastic and deterministic processes to bacterial and fungal assembly across spatial scales and through time, we used 16 s rRNA gene and ITS sequencing in the soil of an emblematic wine-growing region of Italy.Combining null- and neutral-modelling, we found that assembly processes were consistent through time, but bacteria and fungi were governed by different processes. At the within-vineyard scale, deterministic selection and homogenising dispersal dominated bacterial assembly, while neither selection nor dispersal had clear influence over fungal assembly. At the among-vineyard scale, the influence of dispersal limitation increased for both taxonomic groups, but its contribution was much larger for fungal communities. These null-model-based inferences were supported by neutral modelling, which estimated a dispersal rate almost two orders-of-magnitude lower for fungi than bacteria.This indicates that while stochastic processes are important for fungal assembly, bacteria were more influenced by deterministic selection imposed by the biotic and/or abiotic environment. Managing microbes in vineyard soils could thus benefit from strategies that account for dispersal limitation of fungi and the importance of environmental conditions for bacteria. Our results are consistent with theoretical expectations whereby larger individual size and smaller populations can lead to higher levels of stochasticity.

Characterization of 25 full-length S-RNase alleles, including flanking regions, from a pool of resequenced apple cultivars.

KEY MESSAGE: Data obtained from Illumina resequencing of 63 apple cultivars were used to obtain full-length S-RNase sequences using a strategy based on both alignment and de novo assembly of reads. The reproductive biology of apple is regulated by the S-RNase-based gametophytic self-incompatibility system, that is genetically controlled by the single, multi-genic and multi-allelic S locus. Resequencing of apple cultivars provided a huge amount of genetic data, that can be aligned to the reference genome in order to characterize variation to a genome-wide level. However, this approach is not immediately adaptable to the S-locus, due to some peculiar features such as the high degree of polymorphism, lack of colinearity between haplotypes and extensive presence of repetitive elements. In this study we describe a dedicated procedure aimed at characterizing S-RNase alleles from resequenced cultivars. The S-genotype of 63 apple accessions is reported; the full length coding sequence was determined for the 25 S-RNase alleles present in the 63 resequenced cultivars; these included 10 previously incomplete sequences (S 5 , S 6a , S 6b , S 8 , S 11 , S 23 , S 39 , S 46 , S 50 and S 58 ). Moreover, sequence divergence clearly suggests that alleles S 6a and S 6b , proposed to be neutral variants of the same alleles, should be instead considered different specificities. The promoter sequences have also been analyzed, highlighting regions of homology conserved among all the alleles.

Development and validation of the Axiom(®) Apple480K SNP genotyping array.

Cultivated apple (Malus × domestica Borkh.) is one of the most important fruit crops in temperate regions, and has great economic and cultural value. The apple genome is highly heterozygous and has undergone a recent duplication which, combined with a rapid linkage disequilibrium decay, makes it difficult to perform genome-wide association (GWA) studies. Single nucleotide polymorphism arrays offer highly multiplexed assays at a relatively low cost per data point and can be a valid tool for the identification of the markers associated with traits of interest. Here, we describe the development and validation of a 487K SNP Affymetrix Axiom(®) genotyping array for apple and discuss its potential applications. The array has been built from the high-depth resequencing of 63 different cultivars covering most of the genetic diversity in cultivated apple. The SNPs were chosen by applying a focal points approach to enrich genic regions, but also to reach a uniform coverage of non-genic regions. A total of 1324 apple accessions, including the 92 progenies of two mapping populations, have been genotyped with the Axiom(®) Apple480K to assess the effectiveness of the array. A large majority of SNPs (359 994 or 74%) fell in the stringent class of poly high resolution polymorphisms. We also devised a filtering procedure to identify a subset of 275K very robust markers that can be safely used for germplasm surveys in apple. The Axiom(®) Apple480K has now been commercially released both for public and proprietary use and will likely be a reference tool for GWA studies in apple.

Exploration of alternative splicing events in ten different grapevine cultivars.

BACKGROUND: The complex dynamics of gene regulation in plants are still far from being fully understood. Among many factors involved, alternative splicing (AS) in particular is one of the least well documented. For many years, AS has been considered of less relevant in plants, especially when compared to animals, however, since the introduction of next generation sequencing techniques the number of plant genes believed to be alternatively spliced has increased exponentially. RESULTS: Here, we performed a comprehensive high-throughput transcript sequencing of ten different grapevine cultivars, which resulted in the first high coverage atlas of the grape berry transcriptome. We also developed findAS, a software tool for the analysis of alternatively spliced junctions. We demonstrate that at least 44% of multi-exonic genes undergo AS and a large number of low abundance splice variants is present within the 131.622 splice junctions we have annotated from Pinot noir. CONCLUSIONS: Our analysis shows that ~70% of AS events have relatively low expression levels, furthermore alternative splice sites seem to be enriched near the constitutive ones in some extent showing the noise of the splicing mechanisms. However, AS seems to be extensively conserved among the 10 cultivars.

Unfoldome variation upon plant-pathogen interactions: strawberry infection by Colletotrichum acutatum.

Intrinsically disordered proteins (IDPs) are proteins that lack secondary and/or tertiary structure under physiological conditions. These proteins are very abundant in eukaryotic proteomes and play crucial roles in all molecular mechanisms underlying the response to environmental challenges. In plants, different IDPs involved in stress response have been identified and characterized. Nevertheless, a comprehensive evaluation of protein disorder in plant proteomes under abiotic or biotic stresses is not available so far. In the present work the transcriptome dataset of strawberry (Fragaria X ananassa) fruits interacting with the fungal pathogen Colletotrichum acutatum was actualized onto the woodland strawberry (Fragaria vesca) genome. The obtained cDNA sequences were translated into protein sequences, which were subsequently subjected to disorder analysis. The results, providing the first estimation of disorder abundance associated to plant infection, showed that the proteome activated in the strawberry red fruit during the active fungal propagation is remarkably depleted in disorder. On the other hand, in the resistant white fruit, no significant disorder reduction is observed in the proteins expressed in response to fungal infection. Four representative proteins, FvSMP, FvPRKRIP, FvPCD-4 and FvFAM32A-like, predicted as mainly disordered and never experimentally characterized before, were isolated, and the absence of structure was validated at the secondary and tertiary level using circular dichroism and differential scanning fluorimetry. Their quaternary structure was also established using light scattering. The results are discussed considering the role of protein disorder in plant defense.

Identification and characterization of specific motifs in effector proteins of plant parasites using MOnSTER.

Plant pathogens cause billions of dollars of crop loss every year and are a major threat to global food security. Identifying and characterizing pathogens effectors is crucial towards their improved control. Because of their poor sequence conservation, effector identification is challenging, and current methods generate too many candidates without indication for prioritizing experimental studies. In most phyla, effectors contain specific sequence motifs which influence their localization and targets in the plant. Therefore, there is an urgent need to develop bioinformatics tools tailored for pathogen effectors. To circumvent these limitations, we have developed MOnSTER a specific tool that identifies clusters of motifs of protein sequences (CLUMPs). MOnSTER can be fed with motifs identified by de novo tools or from databases such as Pfam and InterProScan. The advantage of MOnSTER is the reduction of motif redundancy by clustering them and associating a score. This score encompasses the physicochemical properties of AAs and the motif occurrences. We built up our method to identify discriminant CLUMPs in oomycetes effectors. Consequently, we applied MOnSTER on plant parasitic nematodes and identified six CLUMPs in about 60% of the known nematode candidate parasitism proteins. Furthermore, we found co-occurrences of CLUMPs with protein domains important for invasion and pathogenicity. The potentiality of this tool goes beyond the effector characterization and can be used to easily cluster motifs and calculate the CLUMP-score on any set of protein sequences.

Prostaglandin D2 synthase controls Schwann cells metabolism.

We previously reported that in the absence of Prostaglandin D2 synthase (L-PGDS) peripheral nerves are hypomyelinated in development and that with aging they present aberrant myelin sheaths. We now demonstrate that L-PGDS expressed in Schwann cells is part of a coordinated program aiming at preserving myelin integrity. In vivo and in vitro lipidomic, metabolomic and transcriptomic analyses confirmed that myelin lipids composition, Schwann cells energetic metabolism and key enzymes controlling these processes are altered in the absence of L-PGDS. Moreover, Schwann cells undergo a metabolic rewiring and turn to acetate as the main energetic source. Further, they produce ketone bodies to ensure glial cell and neuronal survival. Importantly, we demonstrate that all these changes correlate with morphological myelin alterations and describe the first physiological pathway implicated in preserving PNS myelin. Collectively, we posit that myelin lipids serve as a reservoir to provide ketone bodies, which together with acetate represent the adaptive substrates Schwann cells can rely on to sustain the axo-glial unit and preserve the integrity of the PNS.

An evaluation of the PacBio RS platform for sequencing and de novo assembly of a chloroplast genome.

BACKGROUND: Second generation sequencing has permitted detailed sequence characterisation at the whole genome level of a growing number of non-model organisms, but the data produced have short read-lengths and biased genome coverage leading to fragmented genome assemblies. The PacBio RS long-read sequencing platform offers the promise of increased read length and unbiased genome coverage and thus the potential to produce genome sequence data of a finished quality containing fewer gaps and longer contigs. However, these advantages come at a much greater cost per nucleotide and with a perceived increase in error-rate. In this investigation, we evaluated the performance of the PacBio RS sequencing platform through the sequencing and de novo assembly of the Potentilla micrantha chloroplast genome. RESULTS: Following error-correction, a total of 28,638 PacBio RS reads were recovered with a mean read length of 1,902 bp totalling 54,492,250 nucleotides and representing an average depth of coverage of 320× the chloroplast genome. The dataset covered the entire 154,959 bp of the chloroplast genome in a single contig (100% coverage) compared to seven contigs (90.59% coverage) recovered from an Illumina data, and revealed no bias in coverage of GC rich regions. Post-assembly the data were largely concordant with the Illumina data generated and allowed 187 ambiguities in the Illumina data to be resolved. The additional read length also permitted small differences in the two inverted repeat regions to be assigned unambiguously. CONCLUSIONS: This is the first report to our knowledge of a chloroplast genome assembled de novo using PacBio sequence data. The PacBio RS data generated here were assembled into a single large contig spanning the P. micrantha chloroplast genome, with a higher degree of accuracy than an Illumina dataset generated at a much greater depth of coverage, due to longer read lengths and lower GC bias in the data. The results we present suggest PacBio data will be of immense utility for the development of genome sequence assemblies containing fewer unresolved gaps and ambiguities and a significantly smaller number of contigs than could be produced using short-read sequence data alone.

Physiological and Transcriptomic Evaluation of Drought Effect on Own-Rooted and Grafted Grapevine Rootstock (1103P and 101-14MGt).

Grapevines worldwide are grafted onto Vitis spp. rootstocks in order to improve their tolerance to biotic and abiotic stresses. Thus, the response of vines to drought is the result of the interaction between the scion variety and the rootstock genotype. In this work, the responses of genotypes to drought were evaluated on 1103P and 101-14MGt plants, own-rooted and grafted with Cabernet Sauvignon, in three different water deficit conditions (80, 50, and 20% soil water content, SWC). Gas exchange parameters, stem water potential, root and leaf ABA content, and root and leaf transcriptomic response were investigated. Under well-watered conditions, gas exchange and stem water potential were mainly affected by the grafting condition, whereas under sever water deficit they were affected by the rootstock genotype. Under severe stress conditions (20% SWC), 1103P showed an "avoidance" behavior. It reduced stomatal conductance, inhibited photosynthesis, increased ABA content in the roots, and closed the stomata. The 101-14MGt maintained a high photosynthetic rate, limiting the reduction of soil water potential. This behavior results in a "tolerance" strategy. An analysis of the transcriptome showed that most of the differentially expressed genes were detected at 20% SWC, and more significantly in roots than in leaves. A core set of genes has been highlighted on the roots as being related to the root response to drought that are not affected by genotype nor grafting. Genes specifically regulated by grafting and genes specifically regulated by genotype under drought conditions have been identified as well. The 1103P, more than the 101-14MGt, regulated a high number of genes in both own-rooted and grafted conditions. This different regulation revealed that 1103P rootstock readily perceived the water scarcity and rapidly faced the stress, in agreement with its avoidance strategy.

Dissecting the effect of soil on plant phenology and berry transcriptional plasticity in two Italian grapevine varieties (Vitis vinifera L.).

Grapevine embodies a fascinating species as regards phenotypic plasticity and genotype-per-environment interactions. The terroir, namely the set of agri-environmental factors to which a variety is subjected, can influence the phenotype at the physiological, molecular, and biochemical level, representing an important phenomenon connected to the typicality of productions. We investigated the determinants of plasticity by conducting a field-experiment where all terroir variables, except soil, were kept as constant as possible. We isolated the effect of soils collected from different areas, on phenology, physiology, and transcriptional responses of skin and flesh of a red and a white variety of great economic value: Corvina and Glera. Molecular results, together with physio-phenological parameters, suggest a specific effect of soil on grapevine plastic response, highlighting a higher transcriptional plasticity of Glera in respect to Corvina and a marked response of skin compared to flesh. Using a novel statistical approach, we identified clusters of plastic genes subjected to the specific influence of soil. These findings could represent an issue of applicative value, posing the basis for targeted agricultural practices to enhance the desired characteristics for any soil/cultivar combination, to improve vineyards management for a better resource usage and to valorize vineyards uniqueness maximizing the terroir-effect.

Whole genome comparisons of Fragaria, Prunus and Malus reveal different modes of evolution between Rosaceous subfamilies.

BACKGROUND: Rosaceae include numerous economically important and morphologically diverse species. Comparative mapping between the member species in Rosaceae have indicated some level of synteny. Recently the whole genome of three crop species, peach, apple and strawberry, which belong to different genera of the Rosaceae family, have been sequenced, allowing in-depth comparison of these genomes. RESULTS: Our analysis using the whole genome sequences of peach, apple and strawberry identified 1399 orthologous regions between the three genomes, with a mean length of around 100 kb. Each peach chromosome showed major orthology mostly to one strawberry chromosome, but to more than two apple chromosomes, suggesting that the apple genome went through more chromosomal fissions in addition to the whole genome duplication after the divergence of the three genera. However, the distribution of contiguous ancestral regions, identified using the multiple genome rearrangements and ancestors (MGRA) algorithm, suggested that the Fragaria genome went through a greater number of small scale rearrangements compared to the other genomes since they diverged from a common ancestor. Using the contiguous ancestral regions, we reconstructed a hypothetical ancestral genome for the Rosaceae 7 composed of nine chromosomes and propose the evolutionary steps from the ancestral genome to the extant Fragaria, Prunus and Malus genomes. CONCLUSION: Our analysis shows that different modes of evolution may have played major roles in different subfamilies of Rosaceae. The hypothetical ancestral genome of Rosaceae and the evolutionary steps that lead to three different lineages of Rosaceae will facilitate our understanding of plant genome evolution as well as have a practical impact on knowledge transfer among member species of Rosaceae.

Genetic analysis of metabolites in apple fruits indicates an mQTL hotspot for phenolic compounds on linkage group 16.

Apple (Malus×domestica Borkh) is among the main sources of phenolic compounds in the human diet. The genetic basis of the quantitative variations of these potentially beneficial phenolic compounds was investigated. A segregating F1 population was used to map metabolite quantitative trait loci (mQTLs). Untargeted metabolic profiling of peel and flesh tissues of ripe fruits was performed using liquid chromatography-mass spectrometry (LC-MS), resulting in the detection of 418 metabolites in peel and 254 in flesh. In mQTL mapping using MetaNetwork, 669 significant mQTLs were detected: 488 in the peel and 181 in the flesh. Four linkage groups (LGs), LG1, LG8, LG13, and LG16, were found to contain mQTL hotspots, mainly regulating metabolites that belong to the phenylpropanoid pathway. The genetics of annotated metabolites was studied in more detail using MapQTL®. A number of quercetin conjugates had mQTLs on LG1 or LG13. The most important mQTL hotspot with the largest number of metabolites was detected on LG16: mQTLs for 33 peel-related and 17 flesh-related phenolic compounds. Structural genes involved in the phenylpropanoid biosynthetic pathway were located, using the apple genome sequence. The structural gene leucoanthocyanidin reductase (LAR1) was in the mQTL hotspot on LG16, as were seven transcription factor genes. The authors believe that this is the first time that a QTL analysis was performed on such a high number of metabolites in an outbreeding plant species.

Identification of Candidate Genes Associated With Tolerance to Apple Replant Disease by Genome-Wide Transcriptome Analysis.

Apple replant disease (ARD) is a worldwide economic risk in apple cultivation for fruit tree nurseries and fruit growers. Several studies on the reaction of apple plants to ARD are documented but less is known about the genetic mechanisms behind this symptomatology. RNA-seq analysis is a powerful tool for revealing candidate genes that are involved in the molecular responses to biotic stresses in plants. The aim of our work was to find differentially expressed genes in response to ARD in Malus. For this, we compared transcriptome data of the rootstock 'M9' (susceptible) and the wild apple genotype M. ×robusta 5 (Mr5, tolerant) after cultivation in ARD soil and disinfected ARD soil, respectively. When comparing apple plantlets grown in ARD soil to those grown in disinfected ARD soil, 1,206 differentially expressed genes (DEGs) were identified based on a log2 fold change, (LFC) ≥ 1 for up- and ≤ -1 for downregulation (p < 0.05). Subsequent validation revealed a highly significant positive correlation (r = 0.91; p < 0.0001) between RNA-seq and RT-qPCR results indicating a high reliability of the RNA-seq data. PageMan analysis showed that transcripts of genes involved in gibberellic acid (GA) biosynthesis were significantly enriched in the DEG dataset. Most of these GA biosynthesis genes were associated with functions in cell wall stabilization. Further genes were related to detoxification processes. Genes of both groups were expressed significantly higher in Mr5, suggesting that the lower susceptibility to ARD in Mr5 is not due to a single mechanism. These findings contribute to a better insight into ARD response in susceptible and tolerant apple genotypes. However, future research is needed to identify the defense mechanisms, which are most effective for the plant to overcome ARD.

Absence of increased genomic variants in the cyanobacterium Chroococcidiopsis exposed to Mars-like conditions outside the space station.

Despite the increasing interest in using microbial-based technologies to support human space exploration, many unknowns remain not only on bioprocesses but also on microbial survivability and genetic stability under non-Earth conditions. Here the desert cyanobacterium Chroococcidiopsis sp. CCMEE 029 was investigated for robustness of the repair capability of DNA lesions accumulated under Mars-like conditions (UV radiation and atmosphere) simulated in low Earth orbit using the EXPOSE-R2 facility installed outside the International Space Station. Genomic alterations were determined in a space-derivate of Chroococcidiopsis sp. CCMEE 029 obtained upon reactivation on Earth of the space-exposed cells. Comparative analysis of whole-genome sequences showed no increased variant numbers in the space-derivate compared to triplicates of the reference strain maintained on the ground. This result advanced cyanobacteria-based technologies to support human space exploration.

Comparative analysis of rosaceous genomes and the reconstruction of a putative ancestral genome for the family.

BACKGROUND: Comparative genome mapping studies in Rosaceae have been conducted until now by aligning genetic maps within the same genus, or closely related genera and using a limited number of common markers. The growing body of genomics resources and sequence data for both Prunus and Fragaria permits detailed comparisons between these genera and the recently released Malus × domestica genome sequence. RESULTS: We generated a comparative analysis using 806 molecular markers that are anchored genetically to the Prunus and/or Fragaria reference maps, and physically to the Malus genome sequence. Markers in common for Malus and Prunus, and Malus and Fragaria, respectively were 784 and 148. The correspondence between marker positions was high and conserved syntenic blocks were identified among the three genera in the Rosaceae. We reconstructed a proposed ancestral genome for the Rosaceae. CONCLUSIONS: A genome containing nine chromosomes is the most likely candidate for the ancestral Rosaceae progenitor. The number of chromosomal translocations observed between the three genera investigated was low. However, the number of inversions identified among Malus and Prunus was much higher than any reported genome comparisons in plants, suggesting that small inversions have played an important role in the evolution of these two genera or of the Rosaceae.

Resistance to Plasmopara viticola in a grapevine segregating population is associated with stilbenoid accumulation and with specific host transcriptional responses.

BACKGROUND: Downy mildew, caused by the oomycete Plasmopara viticola, is a serious disease in Vitis vinifera, the most commonly cultivated grapevine species. Several wild Vitis species have instead been found to be resistant to this pathogen and have been used as a source to introgress resistance into a V. vinifera background. Stilbenoids represent the major phytoalexins in grapevine, and their toxicity is closely related to the specific compound. The aim of this study was to assess the resistance response to P. viticola of the Merzling × Teroldego cross by profiling the stilbenoid content of the leaves of an entire population and the transcriptome of resistant and susceptible individuals following infection. RESULTS: A three-year analysis of the population's response to artificial inoculation showed that individuals were distributed in nine classes ranging from total resistance to total susceptibility. In addition, quantitative metabolite profiling of stilbenoids in the population, carried out using HPLC-DAD-MS, identified three distinct groups differing according to the concentrations present and the complexity of their profiles. The high producers were characterized by the presence of trans-resveratrol, trans-piceid, trans-pterostilbene and up to thirteen different viniferins, nine of them new in grapevine.Accumulation of these compounds is consistent with a resistant phenotype and suggests that they may contribute to the resistance response.A preliminary transcriptional study using cDNA-AFLP selected a set of genes modulated by the oomycete in a resistant genotype. The expression of this set of genes in resistant and susceptible genotypes of the progeny population was then assessed by comparative microarray analysis.A group of 57 genes was found to be exclusively modulated in the resistant genotype suggesting that they are involved in the grapevine-P. viticola incompatible interaction. Functional annotation of these transcripts revealed that they belong to the categories defense response, photosynthesis, primary and secondary metabolism, signal transduction and transport. CONCLUSIONS: This study reports the results of a combined metabolic and transcriptional profiling of a grapevine population segregating for resistance to P. viticola. Some resistant individuals were identified and further characterized at the molecular level. These results will be valuable to future grapevine breeding programs.

A roadmap for the generation of benchmarking resources for antimicrobial resistance detection using next generation sequencing.

Next Generation Sequencing technologies significantly impact the field of Antimicrobial Resistance (AMR) detection and monitoring, with immediate uses in diagnosis and risk assessment. For this application and in general, considerable challenges remain in demonstrating sufficient trust to act upon the meaningful information produced from raw data, partly because of the reliance on bioinformatics pipelines, which can produce different results and therefore lead to different interpretations. With the constant evolution of the field, it is difficult to identify, harmonise and recommend specific methods for large-scale implementations over time. In this article, we propose to address this challenge through establishing a transparent, performance-based, evaluation approach to provide flexibility in the bioinformatics tools of choice, while demonstrating proficiency in meeting common performance standards. The approach is two-fold: first, a community-driven effort to establish and maintain "live" (dynamic) benchmarking platforms to provide relevant performance metrics, based on different use-cases, that would evolve together with the AMR field; second, agreed and defined datasets to allow the pipelines' implementation, validation, and quality-control over time. Following previous discussions on the main challenges linked to this approach, we provide concrete recommendations and future steps, related to different aspects of the design of benchmarks, such as the selection and the characteristics of the datasets (quality, choice of pathogens and resistances, etc.), the evaluation criteria of the pipelines, and the way these resources should be deployed in the community.

Strategies to produce T-DNA free CRISPRed fruit trees via Agrobacterium tumefaciens stable gene transfer.

Genome editing via CRISPR/Cas9 is a powerful technology, which has been widely applied to improve traits in cereals, vegetables and even fruit trees. For the delivery of CRISPR/Cas9 components into dicotyledonous plants, Agrobacterium tumefaciens mediated gene transfer is still the prevalent method, although editing is often accompanied by the integration of the bacterial T-DNA into the host genome. We assessed two approaches in order to achieve T-DNA excision from the plant genome, minimizing the extent of foreign DNA left behind. The first is based on the Flp/FRT system and the second on Cas9 and synthetic cleavage target sites (CTS) close to T-DNA borders, which are recognized by the sgRNA. Several grapevine and apple lines, transformed with a panel of CRISPR/SpCas9 binary vectors, were regenerated and characterized for T-DNA copy number and for the rate of targeted editing. As detected by an optimized NGS-based sequencing method, trimming at T-DNA borders occurred in 100% of the lines, impairing in most cases the excision. Another observation was the leakage activity of Cas9 which produced pierced and therefore non-functional CTS. Deletions of genomic DNA and presence of filler DNA were also noticed at the junctions between T-DNA and genomic DNA. This study proved that many factors must be considered for designing efficient binary vectors capable of minimizing the presence of exogenous DNA in CRISPRed fruit trees.

Novel Aspects on The Interaction Between Grapevine and Plasmopara viticola: Dual-RNA-Seq Analysis Highlights Gene Expression Dynamics in The Pathogen and The Plant During The Battle For Infection.

Mgaloblishvili, a Vitis vinifera cultivar, exhibits unique resistance traits against Plasmopara viticola, the downy mildew agent. This offers the unique opportunity of exploring the molecular responses in compatible and incompatible plant-pathogen interaction. In this study, whole transcriptomes of Mgaloblishvili, Pinot noir (a V. vinifera susceptible cultivar), and Bianca (a resistant hybrid) leaves, inoculated and non-inoculated with the pathogen, were used to identify P. viticola effector-encoding genes and plant susceptibility/resistance genes. Multiple effector-encoding genes were identified in P. viticola transcriptome, with remarkable expression differences in relation to the inoculated grapevine cultivar. Intriguingly, five apoplastic effectors specifically associated with resistance in V. vinifera. Gene coexpression network analysis identified specific modules and metabolic changes occurring during infection in the three grapevine cultivars. Analysis of these data allowed, for the first time, the detection in V. vinifera of a putative P. viticola susceptibility gene, encoding a LOB domain-containing protein. Finally, the de novo assembly of Mgaloblishvili, Pinot noir, and Bianca transcriptomes and their comparison highlighted novel candidate genes that might be at the basis of the resistant phenotype. These results open the way to functional analysis studies and to new perspectives in molecular breeding of grapevine for resistance to P. viticola.