A cluster of putative resistance genes is associated with a dominant resistance to sunflower broomrape.

KEY MESSAGE: The HaOr5 resistance gene is located in a large genomic insertion containing putative resistance genes and provides resistance to O. cumana, preventing successful connection to the sunflower root vascular system. Orobanche cumana (sunflower broomrape) is a parasitic plant that is part of the Orobanchaceae family and specifically infests sunflower crops. This weed is an obligate parasitic plant that does not carry out photosynthetic activity or develop roots and is fully dependent on its host for its development. It produces thousands of dust-like seeds per plant. It possesses a high spreading ability and has been shown to quickly overcome resistance genes successively introduced by selection in cultivated sunflower varieties. The first part of its life cycle occurs underground. The connection to the sunflower vascular system is essential for parasitic plant survival and development. The HaOr5 gene provides resistance to sunflower broomrape race E by preventing the connection of O. cumana to the root vascular system. We mapped a single position of the HaOr5 gene by quantitative trait locus mapping using two segregating populations. The same location of the HaOr5 gene was identified by genome-wide association. Using a large population of thousands of F2 plants, we restricted the location of the HaOr5 gene to a genomic region of 193 kb. By sequencing the whole genome of the resistant line harboring the major resistance gene HaOr5, we identified a large insertion of a complex genomic region containing a cluster of putative resistance genes.

Evolutionary Analysis of Six Gene Families Part of the Reactive Oxygen Species (ROS) Gene Network in Three Brassicaceae Species.

Climate change is expected to intensify the occurrence of abiotic stress in plants, such as hypoxia and salt stresses, leading to the production of reactive oxygen species (ROS), which need to be effectively managed by various oxido-reductases encoded by the so-called ROS gene network. Here, we studied six oxido-reductases families in three Brassicaceae species, Arabidopsis thaliana as well as Nasturtium officinale and Eutrema salsugineum, which are adapted to hypoxia and salt stress, respectively. Using available and new genomic data, we performed a phylogenomic analysis and compared RNA-seq data to study genomic and transcriptomic adaptations. This comprehensive approach allowed for the gaining of insights into the impact of the adaptation to saline or hypoxia conditions on genome organization (gene gains and losses) and transcriptional regulation. Notably, the comparison of the N. officinale and E. salsugineum genomes to that of A. thaliana highlighted changes in the distribution of ohnologs and homologs, particularly affecting class III peroxidase genes (CIII Prxs). These changes were specific to each gene, to gene families subjected to duplication events and to each species, suggesting distinct evolutionary responses. The analysis of transcriptomic data has allowed for the identification of genes related to stress responses in A. thaliana, and, conversely, to adaptation in N. officinale and E. salsugineum.

An online database for einkorn wheat to aid in gene discovery and functional genomics studies.

Diploid A-genome wheat (einkorn wheat) presents a nutrition-rich option as an ancient grain crop and a resource for the improvement of bread wheat against abiotic and biotic stresses. Realizing the importance of this wheat species, reference-level assemblies of two einkorn wheat accessions were generated (wild and domesticated). This work reports an einkorn genome database that provides an interface to the cereals research community to perform comparative genomics, applied genetics and breeding research. It features queries for annotated genes, the use of a recent genome browser release, and the ability to search for sequence alignments using a modern BLAST interface. Other features include a comparison of reference einkorn assemblies with other wheat cultivars through genomic synteny visualization and an alignment visualization tool for BLAST results. Altogether, this resource will help wheat research and breeding. Database URL  https://wheat.pw.usda.gov/GG3/pangenome.

Einkorn genomics sheds light on history of the oldest domesticated wheat.

Einkorn (Triticum monococcum) was the first domesticated wheat species, and was central to the birth of agriculture and the Neolithic Revolution in the Fertile Crescent around 10,000 years ago1,2. Here we generate and analyse 5.2-Gb genome assemblies for wild and domesticated einkorn, including completely assembled centromeres. Einkorn centromeres are highly dynamic, showing evidence of ancient and recent centromere shifts caused by structural rearrangements. Whole-genome sequencing analysis of a diversity panel uncovered the population structure and evolutionary history of einkorn, revealing complex patterns of hybridizations and introgressions after the dispersal of domesticated einkorn from the Fertile Crescent. We also show that around 1% of the modern bread wheat (Triticum aestivum) A subgenome originates from einkorn. These resources and findings highlight the history of einkorn evolution and provide a basis to accelerate the genomics-assisted improvement of einkorn and bread wheat.

Comparative phylotranscriptomics reveals ancestral and derived root nodule symbiosis programmes.

Symbiotic interactions such as the nitrogen-fixing root nodule symbiosis (RNS) have structured ecosystems during the evolution of life. Here we aimed at reconstructing ancestral and intermediate steps that shaped RNS observed in extant flowering plants. We compared the symbiotic transcriptomic responses of nine host plants, including the mimosoid legume Mimosa pudica for which we assembled a chromosome-level genome. We reconstructed the ancestral RNS transcriptome composed of most known symbiotic genes together with hundreds of novel candidates. Cross-referencing with transcriptomic data in response to experimentally evolved bacterial strains with gradual symbiotic proficiencies, we found the response to bacterial signals, nodule infection, nodule organogenesis and nitrogen fixation to be ancestral. By contrast, the release of symbiosomes was associated with recently evolved genes encoding small proteins in each lineage. We demonstrate that the symbiotic response was mostly in place in the most recent common ancestor of the RNS-forming species more than 90 million years ago.

p57Kip2 acts as a transcriptional corepressor to regulate intestinal stem cell fate and proliferation.

p57Kip2 is a cyclin/CDK inhibitor and a negative regulator of cell proliferation. Here, we report that p57 regulates intestinal stem cell (ISC) fate and proliferation in a CDK-independent manner during intestinal development. In the absence of p57, intestinal crypts exhibit an increased proliferation and an amplification of transit-amplifying cells and of Hopx+ ISCs, which are no longer quiescent, while Lgr5+ ISCs are unaffected. RNA sequencing (RNA-seq) analyses of Hopx+ ISCs show major gene expression changes in the absence of p57. We found that p57 binds to and inhibits the activity of Ascl2, a transcription factor critical for ISC specification and maintenance, by participating in the recruitment of a corepressor complex to Ascl2 target gene promoters. Thus, our data suggest that, during intestinal development, p57 plays a key role in maintaining Hopx+ ISC quiescence and repressing the ISC phenotype outside of the crypt bottom by inhibiting the transcription factor Ascl2 in a CDK-independent manner.

The Rm1 and Rm2 Resistance Genes to Green Peach Aphid (Myzus persicae) Encode the Same TNL Proteins in Peach (Prunus persica L.).

The green peach aphid (GPA), Myzus persicae, is an important pest of the peach crop. Three major dominant resistance genes have already been detected, Rm1 in the Weeping Flower Peach (WFP) clone, Rm2 in the Rubira clone, and Rm3 in the Fen Shouxing clone. In this study, after NGS resequencing of WFP and Rubira, we found that their genomic sequences in the Rm1 and Rm2 region were similar but very different from that of the susceptible reference peach Lovell. We constructed a BAC library for the GPA-resistant WFP and screened four BAC clones to sequence the target region. The new sequence was 61.7 Kb longer than Lovell and was annotated with four different TIR_NBS_LRR genes. Among them, the TNL1 gene was very overexpressed in WFP leaves 24 h after GPA infestation. This gene was also present and expressed in the Rubira clone and had the same sequence as the candidate Rm3 gene, supporting the hypothesis that the three genes share the same origin. In addition, we identified a second TNL, TNL2, located at 35.4 Kb from TNL1 and slightly overexpressed after GPA infestation. Kasp and size molecular markers were designed for use in marker-assisted selection and were validated in a peach segregating population.

Expanding duplication of the testis PHD Finger Protein 7 (PHF7) gene in the chicken genome.

Gene duplications increase genetic and phenotypic diversity and occur in complex genomic regions that are still difficult to sequence and assemble. PHD Finger Protein 7 (PHF7) acts during spermiogenesis for histone-to-histone protamine exchange and is a determinant of male fertility in Drosophila and the mouse. We aimed to explore and characterise in the chicken genome the expanding family of the numerous orthologues of the unique mouse Phf7 gene (highly expressed in the testis), observing the fact that this information is unclear and/or variable according to the versions of databases. We validated nine primer pairs by in silico PCR for their use in screening the chicken bacterial artificial chromosome (BAC) library to produce BAC-derived probes to detect and localise PHF7-like loci by fluorescence in situ hybridisation (FISH). We selected nine BAC that highlighted nine chromosomal regions for a total of 10 distinct PHF7-like loci on five Gallus gallus chromosomes: Chr1 (three loci), Chr2 (two loci), Chr12 (one locus), Chr19 (one locus) and ChrZ (three loci). We sequenced the corresponding BAC by using high-performance PacBio technology. After assembly, we performed annotation with the FGENESH program: there were a total of 116 peptides, including 39 PHF7-like proteins identified by BLASTP. These proteins share a common exon-intron core structure of 8-11 exons. Phylogeny revealed that the duplications occurred first between chromosomal regions and then inside each region. There are other duplicated genes in the identified BAC sequences, suggesting that these genomic regions exhibit a high rate of tandem duplication. We showed that the PHF7 gene, which is highly expressed in the rooster testis, is a highly duplicated gene family in the chicken genome, and this phenomenon probably concerns other bird species.

A genome sequence resource for the genus Passiflora, the genome of the wild diploid species Passiflora organensis.

The genus Passiflora comprises a large group of plants popularly known as passionfruit, much appreciated for their exotic flowers and edible fruits. The species (∼500) are morphologically variable (e.g., growth habit, size, and color of flowers) and are adapted to distinct tropical ecosystems. In this study, we generated the genome of the wild diploid species Passiflora organensis Gardner by adopting a hybrid assembly approach. Passiflora organensis has a small genome of 259 Mbp and a heterozygosity rate of 81%, consistent with its reproductive system. Most of the genome sequences could be integrated into its chromosomes with cytogenomic markers (satellite DNA) as references. The repeated sequences accounted for 58.55% of the total DNA analyzed, and the Tekay lineage was the prevalent retrotransposon. In total, 25,327 coding genes were predicted. Passiflora organensis retains 5,609 singletons and 15,671 gene families. We focused on the genes potentially involved in the locus determining self-incompatibility and the MADS-box gene family, allowing us to infer expansions and contractions within specific subfamilies. Finally, we recovered the organellar DNA. Structural rearrangements and two mitoviruses, besides relics of other mobile elements, were found in the chloroplast and mt-DNA molecules, respectively. This study presents the first draft genome assembly of a wild Passiflora species, providing a valuable sequence resource for genomic and evolutionary studies on the genus, and support for breeding cropped passionfruit species.

Building a cluster of NLR genes conferring resistance to pests and pathogens: the story of the Vat gene cluster in cucurbits.

Most molecularly characterized plant resistance genes (R genes) belong to the nucleotide-binding-site-leucine-rich-repeat (NLR) receptor family and are prone to duplication and transposition with high sequence diversity. In this family, the Vat gene in melon is one of the few R genes known for conferring resistance to insect, i.e., Aphis gossypii, but it has been misassembled and/or mispredicted in the whole genomes of Cucurbits. We examined 14 genomic regions (about 400 kb) derived from long-read assemblies spanning Vat-related genes in Cucumis melo, Cucumis sativus, Citrullus lanatus, Benincasa hispida, Cucurbita argyrosperma, and Momordica charantia. We built the phylogeny of those genes. Investigating the paleohistory of the Vat gene cluster, we revealed a step by step process beginning from a common ancestry in cucurbits older than 50 my. We highlighted Vat exclusively in the Cucumis genera, which diverged about 20 my ago. We then focused on melon, evaluating a minimum duplication rate of Vat in 80 wild and cultivated melon lines using generalist primers; our results suggested that duplication started before melon domestication. The phylogeny of 44 Vat-CDS obtained from 21 melon lines revealed gain and loss of leucine-rich-repeat domains along diversification. Altogether, we revealed the high putative recognition scale offered in melon based on a combination of SNPs, number of leucine-rich-repeat domains within each homolog and number of homologs within each cluster that might jointly confer resistance to a large pest and pathogen spectrum. Based on our findings, we propose possible avenues for breeding programs.

p27 controls Ragulator and mTOR activity in amino acid-deprived cells to regulate the autophagy-lysosomal pathway and coordinate cell cycle and cell growth.

Autophagy is a catabolic process whereby cytoplasmic components are degraded within lysosomes, allowing cells to maintain energy homeostasis during nutrient depletion. Several studies reported that the CDK inhibitor p27Kip1 promotes starvation-induced autophagy by an unknown mechanism. Here we find that p27 controls autophagy via an mTORC1-dependent mechanism in amino acid-deprived cells. During prolonged starvation, a fraction of p27 is recruited to lysosomes, where it interacts with LAMTOR1, a component of the Ragulator complex required for mTORC1 activation. Binding of p27 to LAMTOR1 prevents Ragulator assembly and mTORC1 activation, promoting autophagy. Conversely, p27-/- cells exhibit elevated mTORC1 signalling as well as impaired lysosomal activity and autophagy. This is associated with cytoplasmic sequestration of TFEB, preventing induction of the lysosomal genes required for lysosome function. LAMTOR1 silencing or mTOR inhibition restores autophagy and induces apoptosis in p27-/- cells. Together, these results reveal a direct coordinated regulation between the cell cycle and cell growth machineries.

A Repertory of Rearrangements and the Loss of an Inverted Repeat Region in Passiflora Chloroplast Genomes.

Chloroplast genomes (cpDNA) in angiosperms are usually highly conserved. Although rearrangements have been observed in some lineages, such as Passiflora, the mechanisms that lead to rearrangements are still poorly elucidated. In the present study, we obtained 20 new chloroplast genomes (18 species from the genus Passiflora, and Dilkea retusa and Mitostemma brevifilis from the family Passifloraceae) in order to investigate cpDNA evolutionary history in this group. Passiflora cpDNAs vary in size considerably, with ∼50 kb between shortest and longest. Large inverted repeat (IR) expansions were identified, and at the extreme opposite, the loss of an IR was detected for the first time in Passiflora, a rare event in angiosperms. The loss of an IR region was detected in Passiflora capsularis and Passiflora costaricensis, a species in which occasional biparental chloroplast inheritance has previously been reported. A repertory of rearrangements such as inversions and gene losses were detected, making Passiflora one of the few groups with complex chloroplast genome evolution. We also performed a phylogenomic study based on all the available cp genomes and our analysis implies that there is a need to reconsider the taxonomic classifications of some species in the group.

A receptor-like kinase enhances sunflower resistance to Orobanche cumana.

Orobanche cumana (sunflower broomrape) is an obligate parasitic plant that infects sunflower roots, causing yield losses. Here, by using a map-based cloning strategy, we identified HaOr7-a gene that confers resistance to O. cumana race F-which was found to encode a leucine-rich repeat receptor-like kinase. The complete HAOR7 protein is present in resistant lines of sunflower and prevents O. cumana from connecting to the vascular system of sunflower roots, whereas susceptible lines encode a truncated protein that lacks transmembrane and kinase domains.

µLAS technology for DNA isolation coupled to Cas9-assisted targeting for sequencing and assembly of a 30 kb region in plant genome.

Cas9-assisted targeting of DNA fragments in complex genomes is viewed as an essential strategy to obtain high-quality and continuous sequence data. However, the purity of target loci selected by pulsed-field gel electrophoresis (PFGE) has so far been insufficient to assemble the sequence in one contig. Here, we describe the µLAS technology to capture and purify high molecular weight DNA. First, the technology is optimized to perform high sensitivity DNA profiling with a limit of detection of 20 fg/µl for 50 kb fragments and an analytical time of 50 min. Then, µLAS is operated to isolate a 31.5 kb locus cleaved by Cas9 in the genome of the plant Medicago truncatula. Target purification is validated on a Bacterial Artificial Chromosome plasmid, and subsequently carried out in whole genome with µLAS, PFGE or by combining these techniques. PacBio sequencing shows an enrichment factor of the target sequence of 84 with PFGE alone versus 892 by association of PFGE with µLAS. These performances allow us to sequence and assemble one contig of 29 441 bp with 99% sequence identity to the reference sequence.

Genetic basis and timing of a major mating system shift in Capsella.

A crucial step in the transition from outcrossing to self-fertilization is the loss of genetic self-incompatibility (SI). In the Brassicaceae, SI involves the interaction of female and male specificity components, encoded by the genes SRK and SCR at the self-incompatibility locus (S-locus). Theory predicts that S-linked mutations, and especially dominant mutations in SCR, are likely to contribute to loss of SI. However, few studies have investigated the contribution of dominant mutations to loss of SI in wild plant species. Here, we investigate the genetic basis of loss of SI in the self-fertilizing crucifer species Capsella orientalis, by combining genetic mapping, long-read sequencing of complete S-haplotypes, gene expression analyses and controlled crosses. We show that loss of SI in C. orientalis occurred < 2.6 Mya and maps as a dominant trait to the S-locus. We identify a fixed frameshift deletion in the male specificity gene SCR and confirm loss of male SI specificity. We further identify an S-linked small RNA that is predicted to cause dominance of self-compatibility. Our results agree with predictions on the contribution of dominant S-linked mutations to loss of SI, and thus provide new insights into the molecular basis of mating system transitions.

Cytoplasmic p27Kip1 promotes tumorigenesis via suppression of RhoB activity.

The cell cycle inhibitor p27Kip1 is a tumor suppressor via the inhibition of CDK complexes in the nucleus. However, p27 also plays other functions in the cell and may acquire oncogenic roles when located in the cytoplasm. Activation of oncogenic pathways such as Ras or PI3K/AKT causes the relocalization of p27 in the cytoplasm, where it can promote tumorigenesis by unclear mechanisms. Here, we investigated how cytoplasmic p27 participates in the development of non-small cell lung carcinomas. We provide molecular and genetic evidence that the oncogenic role of p27 is mediated, at least in part, by binding to and inhibiting the GTPase RhoB, which normally acts as a tumor suppressor in the lung. Genetically modified mice revealed that RhoB expression is preferentially lost in tumors in which p27 is absent and maintained in tumors expressing wild-type p27 or p27CK- , a mutant that cannot inhibit CDKs. Moreover, although the absence of RhoB promoted tumorigenesis in p27-/- animals, it had no effect in p27CK- knock-in mice, suggesting that cytoplasmic p27 may act as an oncogene, at least in part, by inhibiting the activity of RhoB. Finally, in a cohort of lung cancer patients, we identified a subset of tumors harboring cytoplasmic p27 in which RhoB expression is maintained and these characteristics were strongly associated with decreased patient survival. Thus, monitoring p27 localization and RhoB levels in non-small cell lung carcinoma patients appears to be a powerful prognostic marker for these tumors. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

p27Kip1 regulates the microtubule bundling activity of PRC1.

Cytokinesis begins in anaphase with the formation of the central spindle. PRC1 is a microtubule associated protein that plays an essential role in central spindle formation by crosslinking antiparallel microtubules. We have identified PRC1 as a novel binding partner for p27Kip1 (p27). p27 is a cyclin-CDK inhibitor that causes cell cycle arrest in G1. However, p27 has also been involved in the regulation of G2/M progression and cytokinesis, as well as of other cellular processes, including actin and microtubule cytoskeleton dynamics. We found that p27 interferes with the ability of PRC1 to bind to microtubules, without affecting PRC1 dimerization or its capacity to interact with other partners such as KIF4. In this way, p27 inhibited microtubule bundling by PRC1 in vitro and prevented the extensive microtubule bundling phenotype caused by PRC1 overexpression in cells in culture. Finally, co-expression of p27 or a p27 mutant that does not bind cyclin-CDKs inhibited multinucleation induced by PRC1 overexpression. Together, our results suggest that p27 may participate in the regulation of mitotic progression in a CDK-independent manner by modulating PRC1 activity.

Ma Orthologous Genes in Prunus spp. Shed Light on a Noteworthy NBS-LRR Cluster Conferring Differential Resistance to Root-Knot Nematodes.

Root-knot nematodes (RKNs) are considerable polyphagous pests that severely challenge plants worldwide and especially perennials. The specific genetic resistance of plants mainly relies on the NBS-LRR genes that are pivotal factors for pathogens control. In Prunus spp., the Ma plum and RMja almond genes possess different spectra for resistance to RKNs. While previous works based on the Ma gene allowed to clone it and to decipher its peculiar TIR-NBS-LRR (TNL) structure, we only knew that the RMja gene mapped on the same chromosome as Ma. We carried out a high-resolution mapping using an almond segregating F2 progeny of 1448 seedlings from resistant (R) and susceptible (S) parental accessions, to locate precisely RMja on the peach genome, the reference sequence for Prunus species. We showed that the RMja gene maps in the Ma resistance cluster and that the Ma ortholog is the best candidate for RMja. This co-localization is a crucial step that opens the way to unravel the molecular determinants involved in the resistance to RKNs. Then we sequenced both almond parental NGS genomes and aligned them onto the RKN susceptible reference peach genome. We produced a BAC library of the R parental accession and, from two overlapping BAC clones, we obtained a 336-kb sequence encompassing the RMja candidate region. Thus, we could benefit from three Ma orthologous regions to investigate their sequence polymorphism, respectively, within plum (complete R spectrum), almond (incomplete R spectrum) and peach (null R spectrum). We showed that the Ma TNL cluster has evolved orthologs with a unique conserved structure comprised of five repeated post-LRR (PL) domains, which contain most polymorphism. In addition to support the Ma and RMja orthologous relationship, our results suggest that the polymorphism contained in the PL sequences might underlie differential resistance interactions with RKNs and an original immune mechanism in woody perennials. Besides, our study illustrates how PL exon duplications and losses shape TNL structure and give rise to atypical PL domain repeats of yet unknown role.

p27Kip1 promotes invadopodia turnover and invasion through the regulation of the PAK1/Cortactin pathway.

p27Kip1 (p27) is a cyclin-CDK inhibitor and negative regulator of cell proliferation. p27 also controls other cellular processes including migration and cytoplasmic p27 can act as an oncogene. Furthermore, cytoplasmic p27 promotes invasion and metastasis, in part by promoting epithelial to mesenchymal transition. Herein, we find that p27 promotes cell invasion by binding to and regulating the activity of Cortactin, a critical regulator of invadopodia formation. p27 localizes to invadopodia and limits their number and activity. p27 promotes the interaction of Cortactin with PAK1. In turn, PAK1 promotes invadopodia turnover by phosphorylating Cortactin, and expression of Cortactin mutants for PAK-targeted sites abolishes p27's effect on invadopodia dynamics. Thus, in absence of p27, cells exhibit increased invadopodia stability due to impaired PAK1-Cortactin interaction, but their invasive capacity is reduced compared to wild-type cells. Overall, we find that p27 directly promotes cell invasion by facilitating invadopodia turnover via the Rac1/PAK1/Cortactin pathway.