NB-LRR Lineage-Specific Equipment Is Sorted Out by Sequence Pattern Adaptation and Domain Segment Shuffling.

The nucleotide-binding and leucine-rich repeat (NB-LRR) genes, also known as resistance (R)-genes, play an important role in the activation of immune responses. In recent years, large-scale studies have been performed to highlight the diversification of plant NB-LRR repertories. It is well known that, to provide new functionalities, NB-LRR sequences are subject to duplication, domain fusions and acquisition and other kinds of mutations. Although some mechanisms that govern NB-LRR protein domain adaptations have been uncovered, to retrace the plant-lineage-specific evolution routes of R protein structure, a multi-genome comparative analysis was performed. This study allowed us to define groups of genes sharing homology relationships across different species. It is worth noting that the most populated groups contained well-characterized R proteins. The arsenal profile of such groups was investigated in five botanical families, including important crop species, to underline specific adaptation signatures. In addition, the dissection of 70 NB domains of well-characterized R-genes revealed the NB core motifs from which the three main R protein classes have been diversified. The structural remodeling of domain segments shaped the specific NB-LRR repertoires observed in each plant species. This analysis provided new evolutionary and functional insights on NB protein domain shuffling. Taken together, such findings improved our understanding of the molecular adaptive selection mechanisms occurring at plant R loci.

Multilevel evolution shapes the function of NB-LRR encoding genes in plant innate immunity.

A sophisticated innate immune system based on diverse pathogen receptor genes (PRGs) evolved in the history of plant life. To reconstruct the direction and magnitude of evolutionary trajectories of a given gene family, it is critical to detect the ancestral signatures. The rearrangement of functional domains made up the diversification found in PRG repertoires. Structural rearrangement of ancient domains mediated the NB-LRR evolutionary path from an initial set of modular proteins. Events such as domain acquisition, sequence modification and temporary or stable associations are prominent among rapidly evolving innate immune receptors. Over time PRGs are continuously shaped by different forces to find their optimal arrangement along the genome. The immune system is controlled by a robust regulatory system that works at different scales. It is important to understand how the PRG interaction network can be adjusted to meet specific needs. The high plasticity of the innate immune system is based on a sophisticated functional architecture and multi-level control. Due to the complexity of interacting with diverse pathogens, multiple defense lines have been organized into interconnected groups. Genomic architecture, gene expression regulation and functional arrangement of PRGs allow the deployment of an appropriate innate immunity response.

Prediction of NB-LRR resistance genes based on full-length sequence homology.

The activation of plant immunity is mediated by resistance (R)-gene receptors, also known as nucleotide-binding leucine-rich repeat (NB-LRR) genes, which in turn trigger the authentic defense response. R-gene identification is a crucial goal for both classic and modern plant breeding strategies for disease resistance. The conventional method identifies NB-LRR genes using a protein motif/domain-based search (PDS) within an automatically predicted gene set of the respective genome assembly. PDS proved to be imprecise since repeat masking prior to automatic genome annotation unwittingly prevented comprehensive NB-LRR gene detection. Furthermore, R-genes have diversified in a species-specific manner, so that NB-LRR gene identification cannot be universally standardized. Here, we present the full-length Homology-based R-gene Prediction (HRP) method for the comprehensive identification and annotation of a genome's R-gene repertoire. Our method has substantially addressed the complex genomic organization of tomato (Solanum lycopersicum) NB-LRR gene loci, proving to be more performant than the well-established RenSeq approach. HRP efficiency was also tested on three differently assembled and annotated Beta sp. genomes. Indeed, HRP identified up to 45% more full-length NB-LRR genes compared to previous approaches. HRP also turned out to be a more refined strategy for R-gene allele mining, testified by the identification of hitherto undiscovered Fom-2 homologs in five Cucurbita sp. genomes. In summary, our high-performance method for full-length NB-LRR gene discovery will propel the identification of novel R-genes towards development of improved cultivars.

PRGdb 4.0: an updated database dedicated to genes involved in plant disease resistance process.

The Plant Resistance Genes database (PRGdb; http://prgdb.org/prgdb4/) has been greatly expanded, keeping pace with the increasing amount of available knowledge and data (sequenced proteomes, cloned genes, public analysis data, etc.). The easy-to-use style of the database website has been maintained, while an updated prediction tool, more data and a new section have been added. This new section will contain plant resistance transcriptomic experiments, providing additional easy-to-access experimental information. DRAGO3, the tool for automatic annotation and prediction of plant resistance genes behind PRGdb, has been improved in both accuracy and sensitivity, leading to more reliable predictions. PRGdb offers 199 reference resistance genes and 586.652 putative resistance genes from 182 sequenced proteomes. Compared to the previous release, PRGdb 4.0 has increased the number of reference resistance genes from 153 to 199, the number of putative resistance genes from 177K from 76 proteomes to 586K from 182 sequenced proteomes. A new section has been created that collects plant-pathogen transcriptomic data for five species of agricultural interest. Thereby, with these improvements and data expansions, PRGdb 4.0 aims to serve as a reference to the plant scientific community and breeders worldwide, helping to further study plant resistance mechanisms that contribute to fighting pathogens.

Genomic analysis of the nomenclatural type strain of the nematode-associated entomopathogenic bacterium Providencia vermicola.

BACKGROUND: Enterobacteria of the genus Providencia are mainly known as opportunistic human pathogens but have been isolated from highly diverse natural environments. The species Providencia vermicola comprises insect pathogenic bacteria carried by entomoparasitic nematodes and is investigated as a possible insect biocontrol agent. The recent publication of several genome sequences from bacteria assigned to this species has given rise to inconsistent preliminary results. RESULTS: The genome of the nematode-derived P. vermicola type strain DSM_17385 has been assembled into a 4.2 Mb sequence comprising 5 scaffolds and 13 contigs. A total of 3969 protein-encoding genes were identified. Multilocus sequence typing with different marker sets revealed that none of the previously published presumed P. vermicola genomes represents this taxonomic species. Comparative genomic analysis has confirmed a close phylogenetic relationship of P. vermicola to the P. rettgeri species complex. P. vermicola DSM_17385 carries a type III secretion system (T3SS-1) with probable function in host cell invasion or intracellular survival. Potentially antibiotic resistance-associated genes comprising numerous efflux pumps and point-mutated house-keeping genes, have been identified across the P. vermicola genome. A single small (3.7 kb) plasmid identified, pPVER1, structurally belongs to the qnrD-type family of fluoroquinolone resistance conferring plasmids that is prominent in Providencia and Proteus bacteria, but lacks the qnrD resistance gene. CONCLUSIONS: The sequence reported represents the first well-supported published genome for the taxonomic species P. vermicola to be used as reference in further comparative genomics studies on Providencia bacteria. Due to a striking difference in the type of injectisome encoded by the respective genomes, P. vermicola might operate a fundamentally different mechanism of entomopathogenicity when compared to insect-pathogenic Providencia sneebia or Providencia burhodogranariea. The complete absence of antibiotic resistance gene carrying plasmids or mobile genetic elements as those causing multi drug resistance phenomena in clinical Providencia strains, is consistent with the invertebrate pathogen P. vermicola being in its natural environment efficiently excluded from the propagation routes of multidrug resistance (MDR) carrying genetic elements operating between human pathogens. Susceptibility to MDR plasmid acquisition will likely become a major criterion in the evaluation of P. vermicola for potential applications in biological pest control.

Tomato genomic prediction for good performance under high-temperature and identification of loci involved in thermotolerance response.

Many studies showed that few degrees above tomato optimum growth temperature threshold can lead to serious loss in production. Therefore, the development of innovative strategies to obtain tomato cultivars with improved yield under high temperature conditions is a main goal both for basic genetic studies and breeding activities. In this paper, a F4 segregating population was phenotypically evaluated for quantitative and qualitative traits under heat stress conditions. Moreover, a genotyping by sequencing (GBS) approach has been employed for building up genomic selection (GS) models both for yield and soluble solid content (SCC). Several parameters, including training population size, composition and marker quality were tested to predict genotype performance under heat stress conditions. A good prediction accuracy for the two analyzed traits (0.729 for yield production and 0.715 for SCC) was obtained. The predicted models improved the genetic gain of selection in the next breeding cycles, suggesting that GS approach is a promising strategy to accelerate breeding for heat tolerance in tomato. Finally, the annotation of SNPs located in gene body regions combined with QTL analysis allowed the identification of five candidates putatively involved in high temperatures response, and the building up of a GS model based on calibrated panel of SNP markers.

Large-scale gene gains and losses molded the NLR defense arsenal during the Cucurbita evolution.

MAIN CONCLUSION: Genome-wide annotation reveals that the gene birth-death process of the Cucurbita R family is associated with a species-specific diversification of TNL and CNL protein classes. The Cucurbitaceae family includes nearly 1000 plant species known universally as cucurbits. Cucurbita genus includes many economically important worldwide crops vulnerable to more than 200 pathogens. Therefore, the identification of pathogen-recognition genes is of utmost importance for this genus. The major class of plant-resistance (R) genes encodes nucleotide-binding site and leucine-rich repeat (NLR) proteins, and is divided into three sub-classes namely, TIR-NB-LRR (TNL), CC-NB-LRR (CNL) and RPW8-NB-LRR (RNL). Although the characterization of the NLR gene family has been carried out in important Cucurbita species, this information is still linked to the availability of sequenced genomes. In this study, we analyzed 40 de novo transcriptomes and 5 genome assemblies, which were explored to investigate the Cucurbita expressed-NLR (eNLR) and NLR repertoires using an ad hoc gene annotation approach. Over 1850 NLR-encoding genes were identified, finely characterized and compared to 96 well-characterized plant R-genes. The maximum likelihood analyses revealed an unusual diversification of CNL/TNL genes and a strong RNL conservation. Indeed, several gene gain and loss events have shaped the Cucurbita NLR family. Finally, to provide a first validation step Cucurbita, eNLRs were explored by real-time PCR analysis. The NLR repertories of the 12 Cucurbita species presented in this paper will be useful to discover novel R-genes.

The Tomato Interspecific NB-LRR Gene Arsenal and Its Impact on Breeding Strategies.

Tomato (Solanum lycopersicum L.) is a model system for studying the molecular basis of resistance in plants. The investigation of evolutionary dynamics of tomato resistance (R)-loci provides unique opportunities for identifying factors that promote or constrain genome evolution. Nucleotide-binding domain and leucine-rich repeat (NB-LRR) receptors belong to one of the most plastic and diversified families. The vast amount of genomic data available for Solanaceae and wild tomato relatives provides unprecedented insights into the patterns and mechanisms of evolution of NB-LRR genes. Comparative analysis remarked a reshuffling of R-islands on chromosomes and a high degree of adaptive diversification in key R-loci induced by species-specific pathogen pressure. Unveiling NB-LRR natural variation in tomato and in other Solanaceae species offers the opportunity to effectively exploit genetic diversity in genomic-driven breeding programs with the aim of identifying and introducing new resistances in tomato cultivars. Within this motivating context, we reviewed the repertoire of NB-LRR genes available for tomato improvement with a special focus on signatures of adaptive processes. This issue is still relevant and not thoroughly investigated. We believe that the discovery of mechanisms involved in the generation of a gene with new resistance functions will bring great benefits to future breeding strategies.

Accelerating Tomato Breeding by Exploiting Genomic Selection Approaches.

Genomic selection (GS) is a predictive approach that was built up to increase the rate of genetic gain per unit of time and reduce the generation interval by utilizing genome-wide markers in breeding programs. It has emerged as a valuable method for improving complex traits that are controlled by many genes with small effects. GS enables the prediction of the breeding value of candidate genotypes for selection. In this work, we address important issues related to GS and its implementation in the plant context with special emphasis on tomato breeding. Genomic constraints and critical parameters affecting the accuracy of prediction such as the number of markers, statistical model, phenotyping and complexity of trait, training population size and composition should be carefully evaluated. The comparison of GS approaches for facilitating the selection of tomato superior genotypes during breeding programs is also discussed. GS applied to tomato breeding has already been shown to be feasible. We illustrated how GS can improve the rate of gain in elite line selection, and descendent and backcross schemes. The GS schemes have begun to be delineated and computer science can provide support for future selection strategies. A new promising breeding framework is beginning to emerge for optimizing tomato improvement procedures.

Inferring RPW8-NLRs's evolution patterns in seed plants: case study in Vitis vinifera.

MAIN CONCLUSION: Genomic and transcriptomic studies in plants and, more in deep, in grapevine reveal that the disease-resistance RNL gene family is highly variable. RNLs (RPW8-NLRs) are a phylogenetically distinct class of nucleotide oligomerization domain (NOD)-like receptors (NLRs) identified in plants. Two RNLs, namely, the NRG1 (N Requirement Gene 1) and the ADR1 (Activated Disease Resistance 1), have been characterized; however, little is known about the RNL evolutionary history in higher plants. To trace the diversification of RNL gene subfamily, we scanned the NLR proteins of 73 plant genomes belonging to 29 taxa, revealing a noticeable diversification across species and within the same genus or botanic family together with a conspicuous expansion in important crop species. To explore the RNL variability in Vitis vinifera and gain information with respect to their structure, evolutionary diversification of five grape genomes ('Aglianico', 'Falanghina', 'Sultanina', 'Tannat', and 'Nebbiolo') has been compared to the reference genome ('Pinot Noir'). The number of RNLs ranged from 6 ('Sultanina') to 14 ('Nebbiolo'), in contrast to the 10 'Pinot Noir' RNLs. The phylogenetic study on grapevine RNLs revealed that all collapsed into NRG1-clade, rather than four. To investigate more in depth the means of intraspecific variability of grape RNL copies, a transcriptomic profiling in response to powdery mildew (PM) infection was carried out through qRT-PCRs and public databases interrogation. The RNL expression variability identified in transcriptome data sets supports the hypothesis of a functional expansion/contraction in grapevine varieties. Although no direct correlations between grapevine PM-resistance and RNL expression was identified, our work can provide good candidates for functional studies able to elucidate the putative "helper" role of RNLs in grape immune signalling.

Alien Domains Shaped the Modular Structure of Plant NLR Proteins.

Plant innate immunity mostly relies on nucleotide-binding (NB) and leucine-rich repeat (LRR) intracellular receptors to detect pathogen-derived molecules and to induce defense responses. A multitaxa reconstruction of NB-domain associations allowed us to identify the first NB-LRR arrangement in the Chlorophyta division of the Viridiplantae. Our analysis points out that the basic NOD-like receptor (NLR) unit emerged in Chlorophytes by horizontal transfer and its diversification started from Toll/interleukin receptor-NB-LRR members. The operon-based genomic structure of Chromochloris zofingiensis NLR copies suggests a functional origin of NLR clusters. Moreover, the transmembrane signatures of NLR proteins in the unicellular alga C. zofingiensis support the hypothesis that the NLR-based immunity system of plants derives from a cell-surface surveillance system. Taken together, our findings suggest that NLRs originated in unicellular algae and may have a common origin with cell-surface LRR receptors.

Deciphering the biological processes underlying tomato biomass production and composition.

The huge amounts of biomass residues, remaining in the field after tomato fruits harvesting, can be utilized to produce bioenergy. A multiple level approach aimed to characterize two Solanum pennellii introgression lines (ILs), with contrasting phenotypes for plant architecture and biomass was carried out. The study of gene expression dynamics, microscopy cell traits and qualitative and quantitative cell wall chemical compounds variation enabled the discovery of key genes and cell processes involved biomass accumulation and composition. Enhanced biomass production observed in IL2-6 line is due to a more effective coordination of chloroplasts and mitochondria energy fluxes. Microscopy analysis revealed a higher number of cells and chloroplasts in leaf epidermis in the high biomass line whilst chemical measurements on the two lines pointed out striking differences in the cell wall composition and organization. Taken together, our findings shed light on the mechanisms underlying the tomato biomass production and processability.

A chromosome-anchored eggplant genome sequence reveals key events in Solanaceae evolution.

With approximately 450 species, spiny Solanum species constitute the largest monophyletic group in the Solanaceae family, but a high-quality genome assembly from this group is presently missing. We obtained a chromosome-anchored genome assembly of eggplant (Solanum melongena), containing 34,916 genes, confirming that the diploid gene number in the Solanaceae is around 35,000. Comparative genomic studies with tomato (S. lycopersicum), potato (S. tuberosum) and pepper (Capsicum annuum) highlighted the rapid evolution of miRNA:mRNA regulatory pairs and R-type defense genes in the Solanaceae, and provided a genomic basis for the lack of steroidal glycoalkaloid compounds in the Capsicum genus. Using parsimony methods, we reconstructed the putative chromosomal complements of the key founders of the main Solanaceae clades and the rearrangements that led to the karyotypes of extant species and their ancestors. From 10% to 15% of the genes present in the four genomes were syntenic paralogs (ohnologs) generated by the pre-γ, γ and T paleopolyploidy events, and were enriched in transcription factors. Our data suggest that the basic gene network controlling fruit ripening is conserved in different Solanaceae clades, and that climacteric fruit ripening involves a differential regulation of relatively few components of this network, including CNR and ethylene biosynthetic genes.

Evolutionary conservation of MLO gene promoter signatures.

BACKGROUND: Powdery mildew (PM) is a widespread fungal disease of plants in temperate climates, causing significant economic losses in agricultural settings. Specific homologs of the MLO gene family are PM susceptibility factors, as their loss-of function results in durable PM resistance (mlo resistance) in several plant species. The role of MLO susceptibility genes in plant-pathogen interactions is still elusive, however it is known that they are strongly upregulated following PM infection. RESULTS: In this study, we investigated the structure of 414 Putative Promoter Regions (PPRs) of MLO genes and highlighted motif and regulatory element patterns related to genomic relationships among species and phylogenetic distance among homologs. A TC box-like motif and a thymine-rich motif were found to be overrepresented in MLO genes transcriptionally upregulated upon infection with PM fungi. As proof of concept, we showed that the expression of a melon (Cucumis melo L.) gene enriched for the motifs above mentioned was strongly upregulated upon infection with the PM fungus Podosphaera xanthii. CONCLUSION: While identifying a candidate MLO susceptibility gene in melon, this study provides insight on the transcriptional control of MLO genes and indicates diagnostic features useful to identify MLO susceptibility genes across species affected by the PM disease.

PRGdb 3.0: a comprehensive platform for prediction and analysis of plant disease resistance genes.

The Plant Resistance Genes database (PRGdb; http://prgdb.org) has been redesigned with a new user interface, new sections, new tools and new data for genetic improvement, allowing easy access not only to the plant science research community but also to breeders who want to improve plant disease resistance. The home page offers an overview of easy-to-read search boxes that streamline data queries and directly show plant species for which data from candidate or cloned genes have been collected. Bulk data files and curated resistance gene annotations are made available for each plant species hosted. The new Gene Model view offers detailed information on each cloned resistance gene structure to highlight shared attributes with other genes. PRGdb 3.0 offers 153 reference resistance genes and 177 072 annotated candidate Pathogen Receptor Genes (PRGs). Compared to the previous release, the number of putative genes has been increased from 106 to 177 K from 76 sequenced Viridiplantae and algae genomes. The DRAGO 2 tool, which automatically annotates and predicts (PRGs) from DNA and amino acid with high accuracy and sensitivity, has been added. BLAST search has been implemented to offer users the opportunity to annotate and compare their own sequences. The improved section on plant diseases displays useful information linked to genes and genomes to connect complementary data and better address specific needs. Through, a revised and enlarged collection of data, the development of new tools and a renewed portal, PRGdb 3.0 engages the plant science community in developing a consensus plan to improve knowledge and strategies to fight diseases that afflict main crops and other plants.

Genome-Editing Technologies for Enhancing Plant Disease Resistance.

One of the greatest challenges for agricultural science in the 21st century is to improve yield stability through the progressive development of superior cultivars. The increasing numbers of infectious plant diseases that are caused by plant-pathogens make it ever more necessary to develop new strategies for plant disease resistance breeding. Targeted genome engineering allows the introduction of precise modifications directly into a commercial variety, offering a viable alternative to traditional breeding methods. Genome editing is a powerful tool for modifying crucial players in the plant immunity system. In this work, we propose and discuss genome-editing strategies and targets for improving resistance to phytopathogens. First of all, we present the opportunities to rewrite the effector-target sequence for avoiding effector-target molecular interaction and also to modify effector-target promoters for increasing the expression of target genes involved in the resistance process. In addition, we describe potential approaches for obtaining synthetic R-genes through genome-editing technologies (GETs). Finally, we illustrate a genome editing flowchart to modify the pathogen recognition sites and engineer an R-gene that mounts resistance to some phylogenetically divergent pathogens. GETs potentially mark the beginning of a new era, in which synthetic biology affords a basis for obtaining a reinforced plant defense system. Nowadays it is conceivable that by modulating the function of the major plant immunity players, we will be able to improve crop performance for a sustainable agriculture.

Plant Innate Immunity Multicomponent Model.

Our understanding of plant-pathogen interactions is making rapid advances in order to address issues of global importance such as improving agricultural productivity and sustainable food security. Innate immunity has evolved in plants, resulting in a wide diversity of defense mechanisms adapted to specific threats. The postulated PTI/ETI model describes two perception layers of plant innate immune system, which belong to a first immunity component of defense response activation. To better describe the sophisticated defense system of plants, we propose a new model of plant immunity. This model considers the plant's ability to distinguish the feeding behavior of their many foes, such as a second component that modulates innate immunity. This hypothesis provides a new viewpoint highlighting the relevance of hormone crosstalk and primary metabolism in regulating plant defense against the different behaviors of pathogens with the intention to stimulate further interest in this research area.

Structure, evolution and functional inference on the Mildew Locus O (MLO) gene family in three cultivated Cucurbitaceae spp.

BACKGROUND: The powdery mildew disease affects thousands of plant species and arguably represents the major fungal threat for many Cucurbitaceae crops, including melon (Cucumis melo L.), watermelon (Citrullus lanatus L.) and zucchini (Cucurbita pepo L.). Several studies revealed that specific members of the Mildew Locus O (MLO) gene family act as powdery mildew susceptibility factors. Indeed, their inactivation, as the result of gene knock-out or knock-down, is associated with a peculiar form of resistance, referred to as mlo resistance. RESULTS: We exploited recently available genomic information to provide a comprehensive overview of the MLO gene family in Cucurbitaceae. We report the identification of 16 MLO homologs in C. melo, 14 in C. lanatus and 18 in C. pepo genomes. Bioinformatic treatment of data allowed phylogenetic inference and the prediction of several ortholog pairs and groups. Comparison with functionally characterized MLO genes and, in C. lanatus, gene expression analysis, resulted in the detection of candidate powdery mildew susceptibility factors. We identified a series of conserved amino acid residues and motifs that are likely to play a major role for the function of MLO proteins. Finally, we performed a codon-based evolutionary analysis indicating a general high level of purifying selection in the three Cucurbitaceae MLO gene families, and the occurrence of regions under diversifying selection in candidate susceptibility factors. CONCLUSIONS: Results of this study may help to address further biological questions concerning the evolution and function of MLO genes. Moreover, data reported here could be conveniently used by breeding research, aiming to select powdery mildew resistant cultivars in Cucurbitaceae.

Genetic variability and evolutionary diversification of membrane ABC transporters in plants.

BACKGROUND: ATP-binding cassette proteins have been recognized as playing a crucial role in the regulation of growth and resistance processes in all kingdoms of life. They have been deeply studied in vertebrates because of their role in drug resistance, but much less is known about ABC superfamily functions in plants. RESULTS: Recently released plant genome sequences allowed us to identify 803 ABC transporters in four vascular plants (Oryza. sativa, Solanum lycopersicum, Solanum tuberosum and Vitis vinifera) and 76 transporters in the green alga Volvox carteri, by comparing them with those reannotated in Arabidopsis thaliana and the yeast Saccharomyces cerevisiae. Retrieved proteins have been phylogenetically analysed to infer orthologous relationships. Most orthologous relationships in the A, D, E and F subfamilies were found, and interesting expansions within the ABCG subfamily were observed and discussed. A high level of purifying selection is acting in the five ABC subfamilies A, B, C, D and E. However, evolutionary rates of recent duplicate genes could influence vascular plant genome diversification. The transcription profiles of ABC genes within tomato organs revealed a broad functional role for some transporters and a more specific activity for others, suggesting the presence of key ABC regulators in tomato. CONCLUSIONS: The findings achieved in this work could contribute to address several biological questions concerning the evolution of the relationship between genomes of different species. Plant ABC protein inventories obtained could be a valuable tool both for basic and applied studies. Indeed, interpolation of the putative role of gene functions can accelerate the discovering of new ABC superfamily members.