A nuclease-dead Cas9-derived tool represses target gene expression.

Manipulation of gene expression is central to understanding gene function, engineering cell behavior, and altering biological traits according to production demands. Nuclease-dead Cas9 (dCas9), a variant of active Cas9, offers a versatile platform for the precise control of genome function without DNA cleavage. Notably, however, an effective and universal dCas9-based transcriptional repression system remains unavailable in plants. The non-canonical histone acetyltransferase TENDRIL-LESS (CsTEN) is responsible for chromatin loosening and histone modification in cucumber (Cucumis sativus). In this study, we engineered a gene regulation tool by fusing TEN and its truncated proteins with dCas9. The full-length dCas9-TEN protein substantially repressed gene expression, with the N-terminal domain identified as the core repression domain. We subsequently validated the specificity and efficacy of this system through both transient infection and genetic transformation in cucumber and Arabidopsis (Arabidopsis thaliana). Electrophoretic mobility shift assay (EMSA) revealed the ability of the N-terminal domain of TEN to bind to chromatin, which may promote target binding of the dCas9 complex and enhance the transcriptional repression effect. Our tool enriches the arsenal of genetic regulation tools available for precision breeding in crops.

CsMLO8/11 are required for full susceptibility of cucumber stem to powdery mildew and interact with CsCRK2 and CsRbohD.

Powdery mildew (PM) is one of the most destructive diseases that threaten cucumber production globally. Efficient breeding of novel PM-resistant cultivars will require a robust understanding of the molecular mechanisms of cucumber resistance against PM. Using a genome-wide association study, we detected a locus significantly correlated with PM resistance in cucumber stem, pm-s5.1. A 1449-bp insertion in the CsMLO8 coding region at the pm-s5.1 locus resulted in enhanced stem PM resistance. Knockout mutants of CsMLO8 and CsMLO11 generated by CRISPR/Cas9 both showed improved PM resistance in the stem, hypocotyl, and leaves, and the double mutant mlo8mlo11 displayed even stronger resistance. We found that reactive oxygen species (ROS) accumulation was higher in the stem of these mutants. Protein interaction assays suggested that CsMLO8 and CsMLO11 could physically interact with CsRbohD and CsCRK2, respectively. Further, we showed that CsMLO8 and CsCRK2 competitively interact with the C-terminus of CsRbohD to affect CsCRK2-CsRbohD module-mediated ROS production during PM defense. These findings provide new insights into the understanding of CsMLO proteins during PM defense responses.

Identification and functional characterization of conserved cis-regulatory elements responsible for early fruit development in cucurbit crops.

A number of cis-regulatory elements (CREs) conserved during evolution have been found to be responsible for phenotypic novelty and variation. Cucurbit crops such as cucumber (Cucumis sativus), watermelon (Citrullus lanatus), melon (Cucumis melo), and squash (Cucurbita maxima) develop fruits from an inferior ovary and share some similar biological processes during fruit development. Whether conserved regulatory sequences play critical roles in fruit development of cucurbit crops remains to be explored. In six well-studied cucurbit species, we identified 392,438 conserved noncoding sequences (CNSs), including 82,756 that are specific to cucurbits, by comparative genomics. Genome-wide profiling of accessible chromatin regions (ACRs) and gene expression patterns mapped 20,865 to 43,204 ACRs and their potential target genes for two fruit tissues at two key developmental stages in six cucurbits. Integrated analysis of CNSs and ACRs revealed 4,431 syntenic orthologous CNSs, including 1,687 cucurbit-specific CNSs that overlap with ACRs that are present in all six cucurbit crops and that may regulate the expression of 757 adjacent orthologous genes. CRISPR mutations targeting two CNSs present in the 1,687 cucurbit-specific sequences resulted in substantially altered fruit shape and gene expression patterns of adjacent NAC1 (NAM, ATAF1/2, and CUC2) and EXT-like (EXTENSIN-like) genes, validating the regulatory roles of these CNSs in fruit development. These results not only provide a number of target CREs for cucurbit crop improvement, but also provide insight into the roles of CREs in plant biology and during evolution.

Natural variation in STAYGREEN contributes to low-temperature tolerance in cucumber.

Low-temperature (LT) stress threatens cucumber production globally; however, the molecular mechanisms underlying LT tolerance in cucumber remain largely unknown. Here, using a genome-wide association study (GWAS), we found a naturally occurring single nucleotide polymorphism (SNP) in the STAYGREEN (CsSGR) coding region at the gLTT5.1 locus associated with LT tolerance. Knockout mutants of CsSGR generated by clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 exhibit enhanced LT tolerance, in particularly, increased chlorophyll (Chl) content and reduced reactive oxygen species (ROS) accumulation in response to LT. Moreover, the C-repeat Binding Factor 1 (CsCBF1) transcription factor can directly activate the expression of CsSGR. We demonstrate that the LT-sensitive haplotype CsSGRHapA , but not the LT-tolerant haplotype CsSGRHapG could interact with NON-YELLOW COLORING 1 (CsNYC1) to mediate Chl degradation. Geographic distribution of the CsSGR haplotypes indicated that the CsSGRHapG was selected in cucumber accessions from high latitudes, potentially contributing to LT tolerance during cucumber cold-adaptation in these regions. CsSGR mutants also showed enhanced tolerance to salinity, water deficit, and Pseudoperonospora cubensis, thus CsSGR is an elite target gene for breeding cucumber varieties with broad-spectrum stress tolerance. Collectively, our findings provide new insights into LT tolerance and will ultimately facilitate cucumber molecular breeding.

Comprehensive dissection of meiotic DNA double-strand breaks and crossovers in cucumber.

Meiotic recombination drives genetic diversity and crop genome optimization. In plant breeding, parents with favorable traits are crossed to create elite varieties. Different hybridizations produce diverse types of segment reshuffling between homologous chromosomes. However, little is known about the factors that cause hybrid-specific changes in crossovers (COs). Here, we constructed 2 F2 populations from crosses between a semiwild and 2 domesticated cucumber (Cucumis sativus) accessions and examined CO events. COs mainly occurred around genes and differed unevenly along chromosomes between the 2 hybrids. Fine-scale CO distributions were suppressed in regions of heterozygous structural variations (SVs) and were accelerated by high sequence polymorphism. C. sativus RADiation sensitive 51A (CsRAD51A) binding, histone H3 lysine 4 trimethylation (H3K4me3) modification, chromatin accessibility, and hypomethylation were positively associated with global CO landscapes and in local DNA double-strand break (DSB) hotspots and genes. The frequency and suppression of COs could be roughly predicted based on multiomic information. Differences in CO events between hybrids could be partially traced to distinct genetic and epigenetic features and were significantly associated with specific DSB hotspots and heterozygous SVs. Our findings identify the genomic and epigenetic features that contribute to CO formation and hybrid-specific divergence in cucumber and provide theoretical support for selecting parental combinations and manipulating recombination events at target genomic regions during plant breeding.

Vegetable biology and breeding in the genomics era.

Vegetable crops provide a rich source of essential nutrients for humanity and represent critical economic values to global rural societies. However, genetic studies of vegetable crops have lagged behind major food crops, such as rice, wheat and maize, thereby limiting the application of molecular breeding. In the past decades, genome sequencing technologies have been increasingly applied in genetic studies and breeding of vegetables. In this review, we recapitulate recent progress on reference genome construction, population genomics and the exploitation of multi-omics datasets in vegetable crops. These advances have enabled an in-depth understanding of their domestication and evolution, and facilitated the genetic dissection of numerous agronomic traits, which jointly expedites the exploitation of state-of-the-art biotechnologies in vegetable breeding. We further provide perspectives of further directions for vegetable genomics and indicate how the ever-increasing omics data could accelerate genetic, biological studies and breeding in vegetable crops.

Architecture design of cucurbit crops for enhanced productivity by a natural allele.

Increasing production efficiency is a top priority in agriculture. Optimal plant architecture is the biological basis of dense planting, high crop yield and labour cost savings, and is thus critical for improving agricultural productivity. In cucurbit crops, most species have elongated internodes, but the path to architecture improvement is still not clear. Here we identified a pumpkin accession with a dominant bushy trait, and found that the associated Bush locus harbours a cucurbit-conserved cis-regulatory element in the 5' untranslated region of a transcription factor gene YABBY1. In cucurbit crops, various B-region deletions enhance the translation of YABBY1, with consequent proportional suppression of stem length in a dose-dependent manner. Depending on different cultivation patterns, the precise deployment of these alleles has significant effects on yield improvement or labour cost saving. Our findings demonstrate that the engineering of the YABBY1 B-region is an efficient strategy to customize plant architecture in cucurbit crops.

The genome of homosporous maidenhair fern sheds light on the euphyllophyte evolution and defences.

Euphyllophytes encompass almost all extant plants, including two sister clades, ferns and seed plants. Decoding genomes of ferns is the key to deep insight into the origin of euphyllophytes and the evolution of seed plants. Here we report a chromosome-level genome assembly of Adiantum capillus-veneris L., a model homosporous fern. This fern genome comprises 30 pseudochromosomes with a size of 4.8-gigabase and a contig N50 length of 16.22 Mb. Gene co-expression network analysis uncovered that homospore development in ferns has relatively high genetic similarities with that of the pollen in seed plants. Analysing fern defence response expands understanding of evolution and diversity in endogenous bioactive jasmonates in plants. Moreover, comparing fern genomes with those of other land plants reveals changes in gene families important for the evolutionary novelties within the euphyllophyte clade. These results lay a foundation for studies on fern genome evolution and function, as well as the origin and evolution of euphyllophytes.

Genotype-Specific Recruitment of Rhizosphere Bacteria From Sandy Loam Soil for Growth Promotion of Cucumis sativus var. hardwickii.

The composition and structure of the rhizosphere microbiome is affected by many factors, including soil type, genotype, and cultivation time of the plant. However, the interaction mechanisms among these factors are largely unclear. We use culture-independent 16S rRNA amplicon sequencing to investigate the rhizosphere bacterial composition and the structure of cultivated cucumber Xintaimici (XT) and wild-type cucumber Cucumis sativus var. hardwickii (HD) in four kinds of soils. We found that soil type, cultivation time, and genotype affected the composition and structure of cucumber rhizosphere bacterial communities. Notably, HD showed better physiological features in sandy soil and sandy loam soil than it did in black soil and farm soil at 50 days post-sowing, which was due to its stronger recruitment ability to Nitrospira, Nocardioides, Bacillus, and Gaiella in sandy soil, and more Tumebacillus, Nitrospira, and Paenibacillus in sandy loam soil. Meanwhile, we also found that HD showed a better recruiting capacity for these bacterial genera than XT in both sandy soil and sandy loam soil. Functional predictions indicated that these bacteria might have had stronger root colonization ability and then promoted the growth of cucumbers by enhancing nitrogen metabolism and active metabolite secretion. In this study, our findings provided a better insight into the relationship between cucumber phenotype, genotype, and the rhizosphere bacterial community, which will offer valuable theoretical references for rhizosphere microbiota studies and its future application in agriculture.

Alkaline α-galactosidase 2 (CsAGA2) plays a pivotal role in mediating source-sink communication in cucumber.

Sugars are necessary for plant growth and fruit development. Cucumber (Cucumis sativus L.) transports sugars, mainly raffinose family oligosaccharides (RFOs), in the vascular bundle. As the dominant sugars in cucumber fruit, glucose and fructose are derived from sucrose, which is the product of RFO hydrolysis by α-galactosidase (α-Gal). Here, we characterized the cucumber alkaline α-galactosidase 2 (CsAGA2) gene and found that CsAGA2 has undergone human selection during cucumber domestication. Further experiments showed that the expression of CsAGA2 increases gradually during fruit development, especially in fruit vasculature. In CsAGA2-RNA interference (RNAi) lines, fruit growth was delayed because of lower hexose production in the peduncle and fruit main vascular bundle (MVB). In contrast, CsAGA2-overexpressing (OE) plants displayed bigger fruits. Functional enrichment analysis of transcriptional data indicated that genes related to sugar metabolism, cell wall metabolism, and hormone signaling were significantly downregulated in the peduncle and fruit MVBs of CsAGA2-RNAi plants. Moreover, downregulation of CsAGA2 also caused negative feedback regulation on source leaves, which was shown by reduced photosynthetic efficiency, fewer plasmodesmata at the surface between mesophyll cell and intermediary cell (IC) or between IC and sieve element, and downregulated gene expression and enzyme activities related to phloem loading, as well as decreased sugar production and exportation from leaves and petioles. The opposite trend was observed in CsAGA2-OE lines. Overall, we conclude that CsAGA2 is essential for cucumber fruit set and development through mediation of sugar communication between sink strength and source activity.

NS encodes an auxin transporter that regulates the 'numerous spines' trait in cucumber (Cucumis sativus) fruit.

Fruit spine is an important agronomic trait in cucumber and the "numerous spines (ns)" cucumber varieties are popular in Europe and West Asia. Although the classical genetic locus of ns was reported more than two decades ago, the NS gene has not been cloned yet. In this study, nine genetic loci for the different densities of fruit spines were identified by a genome-wide association study. Among the nine loci, fsdG2.1 was closely associated with the classical genetic locus ns, which harbors a candidate gene Csa2G264590. Overexpression of Csa2G264590 resulted in lower fruit spine density, and the knockout mutant generated by CRISPR/Cas9 displayed an increased spine density, demonstrating that the Csa2G264590 gene is NS. NS is specifically expressed in the fruit peel and spine. Genetic analysis showed that NS regulates fruit spine development independently of the tuberculate gene, Tu, which regulates spine development on tubercules; the cucumber glabrous mutants csgl1 and csgl3 are epistatic to ns. Furthermore, we found that auxin levels in the fruit peel and spine were significantly lower in the knockout mutant ns-cr. Moreover, RNA-sequencing showed that the plant hormone signal transduction pathway was enriched. Notably, most of the auxin responsive Aux/IAA family genes were downregulated in ns-cr. Haplotype analysis showed that the non-functional haplotype of NS exists exclusively in the Eurasian cucumber backgrounds. Taken together, the cloning of NS gene provides new insights into the regulatory network of fruit spine development.

Targeted creating new mutants with compact plant architecture using CRISPR/Cas9 genome editing by an optimized genetic transformation procedure in cucurbit plants.

Fruits and vegetables in the Cucurbitaceae family contribute greatly to the human diet, for example, cucumber, melon, watermelon and squash. The widespread use of genome editing technologies has greatly accelerated the functional characterization of genes as well as crop improvement. However, most economically important cucurbit plants, including melon and squash, remain recalcitrant to standard Agrobacterium tumefaciens-mediated transformation, which limits the effective use of genome editing technology. In this study, we describe the "optimal infiltration intensity" strategy to establish an efficient genetic transformation system for melon and squash. We harnessed the power of this method to target homologs of the ERECTA family of receptor kinase genes and created alleles resulting in a compact plant architecture with shorter internodes in melon, squash and cucumber. The optimized transformation method presented here allows stable CRISPR/Cas9-mediated mutagenesis and will lay a solid foundation for functional gene manipulation in cucurbit crops.

Deletion of a cyclin-dependent protein kinase inhibitor, CsSMR1, leads to dwarf and determinate growth in cucumber (Cucumis sativus L.).

KEY MESSAGE: A 7.9 kb deletion which contains a cyclin-dependent protein kinase inhibitor leads to determinate growth and dwarf phenotype in cucumber. Plant architecture is a composite character which are mainly defined by shoot branching, internode elongation and shoot determinacy. Ideal architecture tends to increase the yield of plants, just like the case of "Green Revolution" increased by the application of semi-dwarf cereal crop varieties in 1960s. Cucumber (Cucumis sativus L.) is an important vegetable cultivated worldwide, and suitable architecture varieties were selected for different production systems. In this study, we obtained a novel dwarf mutant with strikingly shortened plant height and determinate growth habit. By bulked segregant analysis and map-based cloning, we delimited the dw2 locus to a 56.4 kb region which contain five genes. Among all the variations between WT and dw2 within the 56.4 kb region, a 7.9 kb deletion which resulted in complete deletion of CsaV3_5G035790 in dw2 was co-segregated with the dwarf phenotype. Haplotype analysis and gene expression analysis suggest that CsaV3_5G035790 encoding a cyclin-dependent protein kinase inhibitor (CsSMR1) be the candidate gene responsible for the dwarf phenotype in dw2. RNA-seq analysis shows that several kinesin-like proteins, cyclins and reported organ size regulators are expressed differentially between WT and dw2, which may account for the reduced organ size in dwarf plants. Additionally, the down-regulation of CsSTM and CsWOX9 in dw2 resulted in premature termination of shoot apical meristem development, which eventually reduces the internode number and plant height. Identification and characterization of the CsSMR1 provide a new insight into cucumber architecture modification to be applied to mechanized production system.

Regulation of plant architecture by a new histone acetyltransferase targeting gene bodies.

Axillary meristem development determines both plant architecture and crop yield; this critical process is regulated by the PROLIFERATING CELL FACTORS (TCP) family of transcription factors. Although TCP proteins bind primarily to promoter regions, some also target gene bodies for expression activation. However, the underlying regulatory mechanism remains unknown. Here we show that TEN, a TCP from cucumber (Cucumis sativus L.), controls the identity and mobility of tendrils. Through its C terminus, TEN binds at intragenic enhancers of target genes; its N-terminal domain functions as a non-canonical histone acetyltransferase (HAT) to preferentially act on lysine 56 and 122 of the histone H3 globular domain. This HAT activity is responsible for chromatin loosening and host-gene activation. The N termini of all tested CYCLOIDEA and TEOSINTE BRANCHED 1-like TCP proteins contain an intrinsically disordered region; despite their sequence divergence, they have conserved HAT activity. This study identifies a non-canonical class of HATs and provides a mechanism by which modification at the H3 globular domain is integrated with the transcription process.

A summary of second systemic pulmonary shunt for congenital heart disease with pulmonary hypoxemia.

BACKGROUND: There has been an increasing number of children with congenital heart disease that undergo primary or second systemic-pulmonary shunt, while there are few reports on the second systemic-pulmonary shunt. Therefore, this study summarizes the experience of second systemic-pulmonary shunt for congenital heart disease in our hospital. METHODS AND RESULTS: Sixty-five children with congenital heart disease who underwent systemic-pulmonary shunt for the second time in our hospital were analyzed. At the early stage after the operation, cyanosis improved and SpO2 significantly increased. One patient died in hospital (1.54%) and the causes of death were aggravated atrioventricular regurgitation, low cardiac output syndrome, and liver failure. Early complications occurred in 18 patients (27.7%). All the children were rechecked in our hospital every 3-6 months and the McGoon index significantly increased. CONCLUSION: Systemic-pulmonary artery shunt can promote pulmonary vascular development, improve cyanosis symptoms, and increase the chance of radical treatment in children with pulmonary vascular dysplasia.

Genome-wide Target Mapping Shows Histone Deacetylase Complex1 Regulates Cell Proliferation in Cucumber Fruit.

Histone deacetylase (HDAC) proteins participate in diverse and tissue-specific developmental processes by forming various corepressor complexes with different regulatory subunits. An important HDAC machinery hub, the Histone Deacetylase Complex1 (HDC1) protein, participates in multiple protein-protein interactions and regulates organ size in plants. However, the mechanistic basis for this regulation remains unclear. Here, we identified a cucumber (Cucumis sativus) short-fruit mutant (sf2) with a phenotype that includes repressed cell proliferation. SF2 encodes an HDC1 homolog, and its expression is enriched in meristematic tissues, consistent with a role in regulating cell proliferation through the HDAC complex. A weak sf2 allele impairs HDAC targeting to chromatin, resulting in elevated levels of histone acetylation. Genome-wide mapping revealed that SF2 directly targets and promotes histone deacetylation associated with key genes involved in multiple phytohormone pathways and cell cycle regulation, by either directly repressing or activating their expression. We further show that SF2 controls fruit cell proliferation through targeting the biosynthesis and metabolism of cytokinin and polyamines. Our findings reveal a complex regulatory network of fruit cell proliferation mediated by HDC1 and elucidate patterns of HDC1-mediated regulation of gene expression.

FLOWERING LOCUS T Improves Cucumber Adaptation to Higher Latitudes.

Flowering time plays a crucial role in the geographical adaptation of most crops during domestication. Cucumber (Cucumis sativus) is a major vegetable crop worldwide. From its tropical origin on the southern Asian continent, cucumber has spread over a wide latitudinal cline, but the molecular mechanisms underlying this latitudinal adaptation and the expansion of domesticated cucumber are largely unclear. Here, we report the cloning of two flowering time loci from two distinct cucumber populations and show that two large deletions upstream from FLOWERING LOCUS T (FT) are associated with higher expression of FT and earlier flowering. We determined that the two large deletions are pervasive and occurred independently in Eurasian and East-Asian populations. Nucleotide diversity analysis further revealed that the FT locus region of the cucumber genome contains a signature for a selective sweep during domestication. Our results suggest that large genetic structural variations upstream from FT were selected for and have been important in the geographic spread of cucumber from its tropical origin to higher latitudes.

The wax gourd genomes offer insights into the genetic diversity and ancestral cucurbit karyotype.

The botanical family Cucurbitaceae includes a variety of fruit crops with global or local economic importance. How their genomes evolve and the genetic basis of diversity remain largely unexplored. In this study, we sequence the genome of the wax gourd (Benincasa hispida), which bears giant fruit up to 80 cm in length and weighing over 20 kg. Comparative analyses of six cucurbit genomes reveal that the wax gourd genome represents the most ancestral karyotype, with the predicted ancestral genome having 15 proto-chromosomes. We also resequence 146 lines of diverse germplasm and build a variation map consisting of 16 million variations. Combining population genetics and linkage mapping, we identify a number of regions/genes potentially selected during domestication and improvement, some of which likely contribute to the large fruit size in wax gourds. Our analyses of these data help to understand genome evolution and function in cucurbits.

Genetic Regulation of Ethylene Dosage for Cucumber Fruit Elongation.

Plant organ growth and development are determined by a subtle balance between growth stimulation and inhibition. Fruit size and shape are important quality traits influencing yield and market value; however, the underlying mechanism regulating the balance of fruit growth to achieve final size and shape is not well understood. Here, we report a mechanistic model that governs cucumber (Cucumis sativus) fruit elongation through fine-tuning of ethylene homeostasis. We identified a cucumber mutant that bears short fruits owing to repressed cell division. SF1 (Short Fruit 1) encodes a cucurbit-specific RING-type E3 ligase, and the mutation resulted in its enhanced self-ubiquitination and degradation, but accumulation of ACS2 (1-aminocyclopropane-1-carboxylate synthase 2), a rate-limiting enzyme for ethylene biosynthesis. The overproduction of ethylene contributes to the short-fruit phenotype of sf1 Dysfunction of ACS2 resulted in reduced ethylene production, but still repressed cell division and shorter fruit, suggesting that ethylene is still required for basal fruit elongation. SF1 ubiquitinates and degrades both itself and ACS2 to control ethylene synthesis for dose-dependent effect on cell division and fruit elongation. Our findings reveal the mechanism by which ethylene dosage is regulated for the control of cell division in developing fruit.

CsWRKY46, a WRKY transcription factor from cucumber, confers cold resistance in transgenic-plant by regulating a set of cold-stress responsive genes in an ABA-dependent manner.

Plant WRKY transcription factors are trans-regulatory proteins that are involved in plant immune responses, development and senescence; however, their roles in abiotic stress are still not well understood, especially in the horticultural crop cucumber. In this study, a novel cucumber WRKY gene, CsWRKY46 was cloned and identified, which was up-regulated in response to cold stress and exogenous abscisic acid (ABA) treatment. CsWRKY46 is belonging to group II of the WRKY family, CsWRKY46 was found exclusively in the nucleus, as indicated by a transient expression assay. Yeast one-hybrid assay shown that CsWRKY46 interact with the W-box in the promoter of ABI5. Transgenic Arabidopsis lines over-expressing CsWRKY46, WRK46-OE1 and WRK46-OE5 had higher seedling survival rates upon freezing treatment compared with that of the wild-type. The above over-expression lines also showed much a higher proline accumulation, less electrolyte leakage and lower malondialdehyde (MDA) levels. Furthermore, the CsWRKY46 overexpression lines were hypersensitive to ABA during seed germination, but the seedlings were not. Quantitative RT-PCR analyses revealed that the expression levels of the ABA-responsive transcription factor ABI5 were higher in the WRKY46-OE lines than in wild-type and that the overexpression of CsWRKY46 increased the expression of stress-inducible genes, including RD29A and COR47. Taken together, our results demonstrated that CsWRKY46 from cucumber conferred cold tolerance to transgenic plants and positively regulated the cold signaling pathway in an ABA-dependent manner.