First whole genome assembly and annotation of a European common bean cultivar using PacBio HiFi and Iso-Seq data.

Common bean (Phaseolus vulgaris L.) is the most important grain legume for direct human consumption worldwide. Flageolet bean originates from France and presents typical organoleptic properties, including the remarkable feature of having small pale green colored seeds. Here, we report the whole-genome data, assembly and annotation of the flageolet bean accession 'Flavert'. High molecular weight DNA and RNA were extracted and subjected to long-read sequencing using PacBio Sequel II platform. The genome consisted of 566,238,753 bp assembled in 13 molecules, including 11 chromosomes plus the mitochondrial and chloroplastic genomes. Annotation predicted 29,549 protein coding genes and 6,958 non-coding RNA. This high-quality genome (99.2% BUSCO completeness) represents a valuable data set for further genomic and genetic studies on common bean and more generally on legumes. To our knowledge, this is the first whole-genome sequence of a common bean accession originating from Europe.

Whole Genome Sequences of Nine Xanthomonas Strains Responsible for Common Bacterial Blight of Bean.

We report the complete and circularized genome sequences of nine strains of Xanthomonas phaseoli pv. phaseoli and Xanthomonas citri pv. fuscans, which cause common bacterial blight of bean. These assemblies provide high-quality material for functional and evolutionary studies of these legume pathogens.

Improving Common Bacterial Blight Phenotyping by Using Rub Inoculation and Machine Learning: Cheaper, Better, Faster, Stronger.

Accurate assessment of plant symptoms plays a key role for measuring the impact of pathogens during plant-pathogen interaction. Common bacterial blight caused by Xanthomonas phaseoli pv. phaseoli and X. citri pv. fuscans is a major threat to common bean. The pathogenicity of these bacteria is variable among strains and depends mainly on a type III secretion system and associated type III effectors such as transcription activator-like effectors. Because the impact of a single gene is often small and difficult to detect, a discriminating methodology is required to distinguish the slight phenotype changes induced during the progression of the disease. Here, we compared two different inoculation and symptom assessment methods for their ability to distinguish two tal mutants from their corresponding wild-type strains. Interestingly, rub inoculation of the first leaves combined with symptom assessment by machine learning-based imaging allowed significant distinction between wild-type and mutant strains. By contrast, dip inoculation of first-trifoliate leaves combined with chlorophyll fluorescence imaging did not differentiate the strains. Furthermore, the new method developed here led to the miniaturization of pathogenicity tests and significant time savings.

Densely Packed Tethered Polymer Nanoislands: A Simulation Study.

COordinated Responsive Arrays of Surface-Linked polymer islands (CORALS) allow for the creation of molecular surfaces with novel and switchable properties. Critical components of CORALs are the uniformly distributed islands of densely grafted polymer chains (nanoislands) separated by regions of bare surface. The grafting footprint and separation distances of nanoislands are comparable to that of the constituent polymer chains themselves. Herein, we characterize the structural features of the nanoislands and semiflexible polymers within to better understand this critical constituent of CORALs. We observe different characteristics of grafted semiflexible polymers depending on the polymer island's size and distance from the center of the island. Specifically, the characteristics of the chains at the island periphery are similar to isolated tethered polymer chains (full flexible chains), while chains in the center of the island experience the neighbor effect such as chains in the classic polymer brush. Chains close to the edge of the islands exhibit unique structural features between these two regimes. These results can be used in the rational design of CORALs with specific interfacial characteristics and predictable responses to external stimuli. It is hoped that this the discussion of the different morphologies of the polymers as a function of distance from the edge of the polymer will find applications in a wide variety of systems.

Complete and Circularized Genome Sequences of Three Xanthomonas Strains Pathogenic on Soybean and Alfalfa.

We report the complete and circularized genome sequences of two strains of Xanthomonas citri pv. glycines causing bacterial pustule on soybean and one strain of Xanthomonas euvesicatoria pv. alfalfae causing bacterial leaf and stem spot on alfalfa. These assemblies provide high-quality material for functional and evolutionary studies of these legume pathogens.

Complete and Circularized Genome Sequences of 17 Xanthomonas Strains Responsible for Common Bacterial Blight of Bean.

We report the complete and circularized genome sequences of 17 strains of Xanthomonas citri pv. fuscans and Xanthomonas phaseoli pv. phaseoli, which cause common bacterial blight of bean. These new assemblies combining PacBio and short-read sequencing methods provide high-quality material for studying the evolution of these plant pathogens.

Common bacterial blight of bean: a model of seed transmission and pathological convergence.

BACKGROUND: Xanthomonas citri pv. fuscans (Xcf) and Xanthomonas phaseoli pv. phaseoli (Xpp) are the causal agents of common bacterial blight of bean (CBB), an important disease worldwide that remains difficult to control. These pathogens belong to distinct species within the Xanthomonas genus and have undergone a dynamic evolutionary history including the horizontal transfer of genes encoding factors probably involved in adaptation to and pathogenicity on common bean. Seed transmission is a key point of the CBB disease cycle, favouring both vertical transmission of the pathogen and worldwide distribution of the disease through global seed trade. TAXONOMY: Kingdom: Bacteria; phylum: Proteobacteria; class: Gammaproteobacteria; order: Lysobacterales (also known as Xanthomonadales); family: Lysobacteraceae (also known as Xanthomonadaceae); genus: Xanthomonas; species: X. citri pv. fuscans and X. phaseoli pv. phaseoli (Xcf-Xpp). HOST RANGE: The main host of Xcf-Xpp is the common bean (Phaseolus vulgaris). Lima bean (Phaseolus lunatus) and members of the Vigna genus (Vigna aconitifolia, Vigna angularis, Vigna mungo, Vigna radiata, and Vigna umbellata) are also natural hosts of Xcf-Xpp. Natural occurrence of Xcf-Xpp has been reported for a handful of other legumes such as Calopogonium sp., Pueraria sp., pea (Pisum sativum), Lablab purpureus, Macroptilium lathyroides, and Strophostyles helvola. There are conflicting reports concerning the natural occurrence of CBB agents on tepary bean (Phaseolus acutifolius) and cowpea (Vigna unguiculata subsp. unguiculata). SYMPTOMS: CBB symptoms occur on all aerial parts of beans, that is, seedlings, leaves, stems, pods, and seeds. Symptoms initially appear as water-soaked spots evolving into necrosis on leaves, pustules on pods, and cankers on twigs. In severe infections, defoliation and wilting may occur. DISTRIBUTION: CBB is distributed worldwide, meaning that it is frequently encountered in most places where bean is cultivated in the Americas, Asia, Africa, and Oceania, except for arid tropical areas. Xcf-Xpp are regulated nonquarantine pathogens in Europe and are listed in the A2 list by the European and Mediterranean Plant Protection Organization (EPPO). GENOME: The genome consists of a single circular chromosome plus one to four extrachromosomal plasmids of various sizes, for a total mean size of 5.27 Mb with 64.7% GC content and an average predicted number of 4,181 coding sequences. DISEASE CONTROL: Management of CBB is based on integrated approaches that comprise measures aimed at avoiding Xcf-Xpp introduction through infected seeds, cultural practices to limit Xcf-Xpp survival between host crops, whenever possible the use of tolerant or resistant bean genotypes, and chemical treatments, mainly restricted to copper compounds. The use of pathogen-free seeds is essential in an effective management strategy and requires appropriate sampling, detection, and identification methods. USEFUL WEBSITES: https://gd.eppo.int/taxon/XANTPH, https://gd.eppo.int/taxon/XANTFF, and http://www.cost.eu/COST_Actions/ca/CA16107.

Correction to: Common bean resistance to Xanthomonas is associated with upregulation of the salicylic acid pathway and downregulation of photosynthesis.

An amendment to this paper has been published and can be accessed via the original article.

Common bean resistance to Xanthomonas is associated with upregulation of the salicylic acid pathway and downregulation of photosynthesis.

BACKGROUND: Common bacterial blight (CBB) caused by Xanthomonas phaseoli pv. phaseoli and Xanthomonas citri pv. fuscans is one of the major threats to common bean crops (Phaseolus vulgaris L.). Resistance to CBB is particularly complex as 26 quantitative resistance loci to CBB have been described so far. To date, transcriptomic studies after CBB infection have been very scarce and the molecular mechanisms underlying susceptibility or resistance are largely unknown. RESULTS: We sequenced and annotated the genomes of two common bean genotypes being either resistant (BAT93) or susceptible (JaloEEP558) to CBB. Reciprocal BLASTp analysis led to a list of 20,787 homologs between these genotypes and the common bean reference genome (G19833), which provides a solid dataset for further comparative analyses. RNA-Seq after inoculation with X. phaseoli pv. phaseoli showed that the susceptible genotype initiated a more intense and diverse biological response than the resistant genotype. Resistance was linked to upregulation of the salicylic acid pathway and downregulation of photosynthesis and sugar metabolism, while susceptibility was linked to downregulation of resistance genes and upregulation of the ethylene pathway and of genes involved in cell wall modification. CONCLUSIONS: This study helps better understanding the mechanisms occurring during the early colonization phase of common bean by Xanthomonas and unveils new actors potentially important for resistance and susceptibility to CBB. We discuss the potential link between the pathways induced during bean colonization and genes induced by transcription activator-like effectors (TALEs), as illustrated in other Xanthomonas pathovars.

Studying Peripheral T Cell Homeostasis in Mice: A Concise Technical Review.

For several years, it was believed that the thymus was entirely responsible for maintaining T cell homeostasis. Today, it is well-known that homeostatic peripheral mechanisms are essential in order to maintain T cell numbers and diversity constant in the periphery. Naïve and memory T cells require continual access to self-peptide MHC class I and II molecules and/or cytokines to survive in the periphery. Under normal conditions, homeostatic resources are low, and lymphocytes undergo very slow proliferation and survive. Following T cell depletion, the bioavailability of homeostatic resources is significantly increased, and T cell proliferation is dramatically augmented. The development of lymphopenic mouse models has helped our current understanding of factors involved in the regulation of peripheral T cell homeostasis. In this minireview, we will give a brief overview about basic techniques used to study peripheral T cell homeostasis in mice.

Common Bean Subtelomeres Are Hot Spots of Recombination and Favor Resistance Gene Evolution.

Subtelomeres of most eukaryotes contain fast-evolving genes usually involved in adaptive processes. In common bean (Phaseolus vulgaris), the Co-2 anthracnose resistance (R) locus corresponds to a cluster of nucleotide-binding-site leucine-rich-repeat (NL) encoding sequences, the prevalent class of plant R genes. To study the recent evolution of this R gene cluster, we used a combination of sequence, genetic and cytogenetic comparative analyses between common bean genotypes from two distinct gene pools (Andean and Mesoamerican) that diverged 0.165 million years ago. Co-2 is a large subtelomeric cluster on chromosome 11 comprising from 32 (Mesoamerican) to 52 (Andean) NL sequences embedded within khipu satellite repeats. Since the recent split between Andean and Mesoamerican gene pools, the Co-2 cluster has experienced numerous gene-pool specific NL losses, leading to distinct NL repertoires. The high proportion of solo-LTR retrotransposons indicates that the Co-2 cluster is located in a hot spot of unequal intra-strand homologous recombination. Furthermore, we observe large segmental duplications involving both Non-Homologous End Joining and Homologous Recombination double-strand break repair pathways. Finally, the identification of a Mesoamerican-specific subtelomeric sequence reveals frequent interchromosomal recombinations between common bean subtelomeres. Altogether, our results highlight that common bean subtelomeres are hot spots of recombination and favor the rapid evolution of R genes. We propose that chromosome ends could act as R gene incubators in many plant genomes.

Horizontal gene transfer plays a major role in the pathological convergence of Xanthomonas lineages on common bean.

BACKGROUND: Host specialization is a hallmark of numerous plant pathogens including bacteria, fungi, oomycetes and viruses. Yet, the molecular and evolutionary bases of host specificity are poorly understood. In some cases, pathological convergence is observed for individuals belonging to distant phylogenetic clades. This is the case for Xanthomonas strains responsible for common bacterial blight of bean, spread across four genetic lineages. All the strains from these four lineages converged for pathogenicity on common bean, implying possible gene convergences and/or sharing of a common arsenal of genes conferring the ability to infect common bean. RESULTS: To search for genes involved in common bean specificity, we used a combination of whole-genome analyses without a priori, including a genome scan based on k-mer search. Analysis of 72 genomes from a collection of Xanthomonas pathovars unveiled 115 genes bearing DNA sequences specific to strains responsible for common bacterial blight, including 20 genes located on a plasmid. Of these 115 genes, 88 were involved in successive events of horizontal gene transfers among the four genetic lineages, and 44 contained nonsynonymous polymorphisms unique to the causal agents of common bacterial blight. CONCLUSIONS: Our study revealed that host specificity of common bacterial blight agents is associated with a combination of horizontal transfers of genes, and highlights the role of plasmids in these horizontal transfers.

Xanthomonas adaptation to common bean is associated with horizontal transfers of genes encoding TAL effectors.

BACKGROUND: Common bacterial blight is a devastating bacterial disease of common bean (Phaseolus vulgaris) caused by Xanthomonas citri pv. fuscans and Xanthomonas phaseoli pv. phaseoli. These phylogenetically distant strains are able to cause similar symptoms on common bean, suggesting that they have acquired common genetic determinants of adaptation to common bean. Transcription Activator-Like (TAL) effectors are bacterial type III effectors that are able to induce the expression of host genes to promote infection or resistance. Their capacity to bind to a specific host DNA sequence suggests that they are potential candidates for host adaption. RESULTS: To study the diversity of tal genes from Xanthomonas strains responsible for common bacterial blight of bean, whole genome sequences of 17 strains representing the diversity of X. citri pv. fuscans and X. phaseoli pv. phaseoli were obtained by single molecule real time sequencing. Analysis of these genomes revealed the existence of four tal genes named tal23A, tal20F, tal18G and tal18H, respectively. While tal20F and tal18G were chromosomic, tal23A and tal18H were carried on plasmids and shared between phylogenetically distant strains, therefore suggesting recent horizontal transfers of these genes between X. citri pv. fuscans and X. phaseoli pv. phaseoli strains. Strikingly, tal23A was present in all strains studied, suggesting that it played an important role in adaptation to common bean. In silico predictions of TAL effectors targets in the common bean genome suggested that TAL effectors shared by X. citri pv. fuscans and X. phaseoli pv. phaseoli strains target the promoters of genes of similar functions. This could be a trace of convergent evolution among TAL effectors from different phylogenetic groups, and comforts the hypothesis that TAL effectors have been implied in the adaptation to common bean. CONCLUSIONS: Altogether, our results favour a model where plasmidic TAL effectors are able to contribute to host adaptation by being horizontally transferred between distant lineages.

First Complete Genome Sequences of Xanthomonas citri pv. vignicola Strains CFBP7111, CFBP7112, and CFBP7113 Obtained Using Long-Read Technology.

Xanthomonas citri pv. vignicola strains cause bacterial blight of the legume crop cowpea. We report whole-genome sequences of three X. citri pv. vignicola strains obtained using PacBio single-molecule real-time sequencing. Such genomic data provide new information on pathogenicity factors, such as transcription activator-like effectors.

Using Ecology, Physiology, and Genomics to Understand Host Specificity in Xanthomonas.

How pathogens coevolve with and adapt to their hosts are critical to understanding how host jumps and/or acquisition of novel traits can lead to new disease emergences. The Xanthomonas genus includes Gram-negative plant-pathogenic bacteria that collectively infect a broad range of crops and wild plant species. However, individual Xanthomonas strains usually cause disease on only a few plant species and are highly adapted to their hosts, making them pertinent models to study host specificity. This review summarizes our current understanding of the molecular basis of host specificity in the Xanthomonas genus, with a particular focus on the ecology, physiology, and pathogenicity of the bacterium. Despite our limited understanding of the basis of host specificity, type III effectors, microbe-associated molecular patterns, lipopolysaccharides, transcriptional regulators, and chemotactic sensors emerge as key determinants for shaping host specificity.

Immunity to plant pathogens and iron homeostasis.

Iron is essential for metabolic processes in most living organisms. Pathogens and their hosts often compete for the acquisition of this nutrient. However, iron can catalyze the formation of deleterious reactive oxygen species. Hosts may use iron to increase local oxidative stress in defense responses against pathogens. Due to this duality, iron plays a complex role in plant-pathogen interactions. Plant defenses against pathogens and plant response to iron deficiency share several features, such as secretion of phenolic compounds, and use common hormone signaling pathways. Moreover, fine tuning of iron localization during infection involves genes coding iron transport and iron storage proteins, which have been shown to contribute to immunity. The influence of the plant iron status on the outcome of a given pathogen attack is strongly dependent on the nature of the pathogen infection strategy and on the host species. Microbial siderophores emerged as important factors as they have the ability to trigger plant defense responses. Depending on the plant species, siderophore perception can be mediated by their strong iron scavenging capacity or possibly via specific recognition as pathogen associated molecular patterns. This review highlights that iron has a key role in several plant-pathogen interactions by modulating immunity.

Scavenging iron: a novel mechanism of plant immunity activation by microbial siderophores.

Siderophores are specific ferric iron chelators synthesized by virtually all microorganisms in response to iron deficiency. We have previously shown that they promote infection by the phytopathogenic enterobacteria Dickeya dadantii and Erwinia amylovora. Siderophores also have the ability to activate plant immunity. We have used complete Arabidopsis transcriptome microarrays to investigate the global transcriptional modifications in roots and leaves of Arabidopsis (Arabidopsis thaliana) plants after leaf treatment with the siderophore deferrioxamine (DFO). Physiological relevance of these transcriptional modifications was validated experimentally. Immunity and heavy-metal homeostasis were the major processes affected by DFO. These two physiological responses could be activated by a synthetic iron chelator ethylenediamine-di(o-hydroxyphenylacetic) acid, indicating that siderophores eliciting activities rely on their strong iron-chelating capacity. DFO was able to protect Arabidopsis against the pathogenic bacterium Pseudomonas syringae pv tomato DC3000. Siderophore treatment caused local modifications of iron distribution in leaf cells visible by ferrocyanide and diaminobenzidine-H2O2 staining. Metal quantifications showed that DFO causes a transient iron and zinc uptake at the root level, which is presumably mediated by the metal transporter iron regulated transporter1 (IRT1). Defense gene expression and callose deposition in response to DFO were compromised in an irt1 mutant. Consistently, plant susceptibility to D. dadantii was increased in the irt1 mutant. Our work shows that iron scavenging is a unique mechanism of immunity activation in plants. It highlights the strong relationship between heavy-metal homeostasis and immunity.

The Subtelomeric khipu Satellite Repeat from Phaseolus vulgaris: Lessons Learned from the Genome Analysis of the Andean Genotype G19833.

Subtelomeric regions in eukaryotic organisms are known for harboring species-specific tandemly repeated satellite sequences. However, studies on the molecular organization and evolution of subtelomeric repeats are scarce, especially in plants. Khipu is a satellite DNA of 528-bp repeat unit, specific of the Phaseolus genus, with a subtelomeric distribution in common bean, P. vulgaris. To investigate the genomic organization and the evolution of khipu, we performed genome-wide analysis on the complete genome sequence of the common bean genotype G19833. We identified 2,460 khipu units located at most distal ends of the sequenced regions. Khipu units are arranged in discrete blocks of 2-55 copies and are heterogeneously distributed among the different chromosome ends of G19833 (from 0 to 555 khipus units per chromosome arm). Phylogenetically related khipu units are spread between numerous chromosome ends, suggesting frequent exchanges between non-homologous subtelomeres. However, most subclades contain numerous khipu units from only one or few chromosome ends indicating that local duplication is also driving khipu expansion. Unexpectedly, we also identified 81 khipu units located at centromeres. All the centromeric khipu units belong to a single divergent clade also comprised of a few units from several subtelomeres, suggesting that a few sequence exchanges between centromeres and subtelomeres took place in the common bean genome. The divergence and low copy number of these centromeric units from the subtelomeric units could explain why they were not detected by FISH (Fluorescence in situ Hybridization) although it can not be excluded that these centromeric units may have resulted from errors in the pseudomolecule assembly. Altogether our data highlight extensive sequence exchanges in subtelomeres between non-homologous chromosomes in common bean and confirm that subtelomeres represent one of the most dynamic and rapidly evolving regions in eukaryotic genomes.

Identification and characterization of functional centromeres of the common bean.

In higher eukaryotes, centromeres are typically composed of megabase-sized arrays of satellite repeats that evolve rapidly and homogenize within a species' genome. Despite the importance of centromeres, our knowledge is limited to a few model species. We conducted a comprehensive analysis of common bean (Phaseolus vulgaris) centromeric satellite DNA using genomic data, fluorescence in situ hybridization (FISH), immunofluorescence and chromatin immunoprecipitation (ChIP). Two unrelated centromere-specific satellite repeats, CentPv1 and CentPv2, and the common bean centromere-specific histone H3 (PvCENH3) were identified. FISH showed that CentPv1 and CentPv2 are predominantly located at subsets of eight and three centromeres, respectively. Immunofluorescence- and ChIP-based assays demonstrated the functional significance of CentPv1 and CentPv2 at centromeres. Genomic analysis revealed several interesting features of CentPv1 and CentPv2: (i) CentPv1 is organized into an higher-order repeat structure, named Nazca, of 528 bp, whereas CentPv2 is composed of tandemly organized monomers; (ii) CentPv1 and CentPv2 have undergone chromosome-specific homogenization; and (iii) CentPv1 and CentPv2 are not likely to be commingled in the genome. These findings suggest that two distinct sets of centromere sequences have evolved independently within the common bean genome, and provide insight into centromere satellite evolution.

Evolution of a complex disease resistance gene cluster in diploid Phaseolus and tetraploid Glycine.

We used a comparative genomics approach to investigate the evolution of a complex nucleotide-binding (NB)-leucine-rich repeat (LRR) gene cluster found in soybean (Glycine max) and common bean (Phaseolus vulgaris) that is associated with several disease resistance (R) genes of known function, including Rpg1b (for Resistance to Pseudomonas glycinea1b), an R gene effective against specific races of bacterial blight. Analysis of domains revealed that the amino-terminal coiled-coil (CC) domain, central nucleotide-binding domain (NB-ARC [for APAF1, Resistance genes, and CED4]), and carboxyl-terminal LRR domain have undergone distinct evolutionary paths. Sequence exchanges within the NB-ARC domain were rare. In contrast, interparalogue exchanges involving the CC and LRR domains were common, consistent with both of these regions coevolving with pathogens. Residues under positive selection were overrepresented within the predicted solvent-exposed face of the LRR domain, although several also were detected within the CC and NB-ARC domains. Superimposition of these latter residues onto predicted tertiary structures revealed that the majority are located on the surface, suggestive of a role in interactions with other domains or proteins. Following polyploidy in the Glycine lineage, NB-LRR genes have been preferentially lost from one of the duplicated chromosomes (homeologues found in soybean), and there has been partitioning of NB-LRR clades between the two homeologues. The single orthologous region in common bean contains approximately the same number of paralogues as found in the two soybean homeologues combined. We conclude that while polyploidization in Glycine has not driven a stable increase in family size for NB-LRR genes, it has generated two recombinationally isolated clusters, one of which appears to be in the process of decay.