Repeat turnover meets stable chromosomes: repetitive DNA sequences mark speciation and gene pool boundaries in sugar beet and wild beets.

Sugar beet and its wild relatives share a base chromosome number of nine and similar chromosome morphologies. Yet, interspecific breeding is impeded by chromosome and sequence divergence that is still not fully understood. Since repetitive DNAs are among the fastest evolving parts of the genome, we investigated, if repeatome innovations and losses are linked to chromosomal differentiation and speciation. We traced genome and chromosome-wide evolution across 13 beet species comprising all sections of the genera Beta and Patellifolia. For this, we combined short and long read sequencing, flow cytometry, and cytogenetics to build a comprehensive framework that spans the complete scale from DNA to chromosome to genome. Genome sizes and repeat profiles reflect the separation into three gene pools with contrasting evolutionary patterns. Among all repeats, satellite DNAs harbor most genomic variability, leading to fundamentally different centromere architectures, ranging from chromosomal uniformity in Beta and Patellifolia to the formation of patchwork chromosomes in Corollinae/Nanae. We show that repetitive DNAs are causal for the genome expansions and contractions across the beet genera, providing insights into the genomic underpinnings of beet speciation. Satellite DNAs in particular vary considerably between beet genomes, leading to the evolution of distinct chromosomal setups in the three gene pools, likely contributing to the barriers in beet breeding. Thus, with their isokaryotypic chromosome sets, beet genomes present an ideal system for studying the link between repeats, genomic variability, and chromosomal differentiation and provide a theoretical fundament for understanding barriers in any crop breeding effort.

Sugar beet PMT5a and STP13 carriers suitable for proton-driven plasma membrane sucrose and glucose import in taproots.

Sugar beet (Beta vulgaris) is the major sugar-producing crop in Europe and Northern America, as the taproot stores sucrose at a concentration of around 20%. Genome sequence analysis together with biochemical and electrophysiological approaches led to the identification and characterization of the TST sucrose transporter driving vacuolar sugar accumulation in the taproot. However, the sugar transporters mediating sucrose uptake across the plasma membrane of taproot parenchyma cells remained unknown. As with glucose, sucrose stimulation of taproot parenchyma cells caused inward proton fluxes and plasma membrane depolarization, indicating a sugar/proton symport mechanism. To decipher the nature of the corresponding proton-driven sugar transporters, we performed taproot transcriptomic profiling and identified the cold-induced PMT5a and STP13 transporters. When expressed in Xenopus laevis oocytes, BvPMT5a was characterized as a voltage- and H+-driven low-affinity glucose transporter, which does not transport sucrose. In contrast, BvSTP13 operated as a high-affinity H+/sugar symporter, transporting glucose better than sucrose, and being more cold-tolerant than BvPMT5a. Modeling of the BvSTP13 structure with bound mono- and disaccharides suggests plasticity of the binding cleft to accommodate the different saccharides. The identification of BvPMT5a and BvSTP13 as taproot sugar transporters could improve breeding of sugar beet to provide a sustainable energy crop.

BvCGT1-mediated differential distribution of flavonoid C-glycosides contributes to plant's response to UV-B stress.

C-glycosides are a predominant class of flavonoids that demonstrate diverse medical properties and plant physiological functions. The chemical stability, structural diversity, and differential aboveground distribution of these compounds in plants make them ideal protectants. However, little is known about the transcriptional regulatory mechanisms that play these diverse roles in plant physiology. In this study, chard was selected from 69 families for its significantly different flavonoid C-glycosides distributions between the aboveground and underground parts to investigate the role and regulatory mechanism of flavonoid C-glycosides in plants. Our results indicate that flavonoid C-glycosides are affected by various stressors, especially UV-B. Through cloning and validation of key biosynthetic genes of flavonoid C-glycosides in chard (BvCGT1), we observed significant effects induced by UV-B radiation. This finding was further confirmed by resistance testing in BvCGT1 silenced chard lines and in Arabidopsis plants with BvCGT1 overexpression. Yeast one-hybrid and dual-luciferase assays were employed to determine the underlying regulatory mechanisms of BvCGT1 in withstanding UV-B stress. These results indicate a potential regulatory role of BvDof8 and BvDof13 in modulating flavonoid C-glycosides content, through their influence on BvCGT1. In conclusion, we have effectively demonstrated the regulation of BvCGT1 by BvDof8 and BvDof13, highlighting their crucial role in plant adaptation to UV-B radiation. Additionally, we have outlined a comprehensive transcriptional regulatory network involving BvDof8 and BvDof13 in response to UV-B radiation.

Debottlenecking the L-DOPA 4,5-dioxygenase step with enhanced tyrosine supply boosts betalain production in Nicotiana benthamiana.

Synthetic biology provides emerging tools to produce valuable compounds in plant hosts as sustainable chemical production platforms. However, little is known about how supply and utilization of precursors is coordinated at the interface of plant primary and specialized metabolism, limiting our ability to efficiently produce high levels of target specialized metabolites in plants. L-Tyrosine is an aromatic amino acid precursor of diverse plant natural products including betalain pigments, which are used as the major natural food red colorants and more recently a visual marker for plant transformation. Here, we studied the impact of enhanced L-tyrosine supply on the production of betalain pigments by expressing arogenate dehydrogenase (TyrA) from table beet (Beta vulgaris, BvTyrAα), which has relaxed feedback inhibition by L-tyrosine. Unexpectedly, betalain levels were reduced when BvTyrAα was coexpressed with the betalain pathway genes in Nicotiana benthamiana leaves; L-tyrosine and 3,4-dihydroxy-L-phenylalanine (L-DOPA) levels were drastically elevated but not efficiently converted to betalains. An additional expression of L-DOPA 4,5-dioxygenase (DODA), but not CYP76AD1 or cyclo-DOPA 5-O-glucosyltransferase, together with BvTyrAα and the betalain pathway, drastically enhanced betalain production, indicating that DODA is a major rate-limiting step of betalain biosynthesis in this system. Learning from this initial test and further debottlenecking the DODA step maximized betalain yield to an equivalent or higher level than that in table beet. Our data suggest that balancing between enhanced supply ("push") and effective utilization ("pull") of precursor by alleviating a bottleneck step is critical in successful plant synthetic biology to produce high levels of target compounds.

Vernalization promotes bolting in sugar beet by inhibiting the transcriptional repressors of BvGI.

Sugar beet (Beta vulgaris L.), a biennial sugar crop, contributes about 16% of the world's sugar production. The transition from vegetative growth, during which sugar accumulated in beet, to reproductive growth, during which sugar exhausted in beet, is determined by vernalization and photoperiod. GIGANTEA (GI) is a key photoperiodic flowering gene that is induced by vernalization in sugar beet. To identify the upstream regulatory factors of BvGI, candidate transcription factors (TF) that were co-expressed with BvGI and could bind to the BvGI promoter were screened based on weighted gene co-expression network analysis (WGCNA) and TF binding site prediction. Subsequently, their transcriptional regulatory role on the BvGI was validated through subcellular localization, dual-luciferase assays and yeast transformation tests. A total of 7,586 differentially expressed genes were identified after vernalization and divided into 18 co-expression modules by WGCNA, of which one (MEcyan) and two (MEdarkorange2 and MEmidnightblue) modules were positively and negatively correlated with the expression of BvGI, respectively. TF binding site predictions using PlantTFDB enabled the screening of BvLHY, BvTCP4 and BvCRF4 as candidate TFs that negatively regulated the expression of BvGI by affecting its transcription. Subcellular localization showed that BvLHY, BvTCP4 and BvCRF4 were localized to the nucleus. The results of dual-luciferase assays and yeast transformation tests showed that the relative luciferase activity and expression of HIS3 was reduced in the BvLHY, BvTCP4 and BvCRF4 transformants, which suggested that the three TFs inhibited the BvGI promoter. In addition, real-time quantitative reverse transcription PCR showed that BvLHY and BvTCP4 exhibited rhythmic expression characteristics similar to that of BvGI, while BvCRF4 did not. Our results revealed that vernalization crosstalked with the photoperiod pathway to initiate bolting in sugar beet by inhibiting the transcriptional repressors of BvGI.

Insights into the molecular basis of beet curly top resistance in sugar beet through a transcriptomic approach at the early stage of symptom development.

Curly top disease caused by Beet curly top virus (BCTV) is a limiting factor for sugar beet production. The most economical and sustainable control of BCTV in sugar beet would be via the growth of resistant cultivars, although most commercial cultivars possess only low-to-moderate quantitative resistance. A double haploid line (KDH13) showed a high level of resistance to BCTV infection. However, the mechanism of resistance and response of this line to BCTV infection is unknown. Here, we tested the response of this line to both local and systemic BCTV infections. The virus replicated at a high level in locally infected tissue but lower than in susceptible KDH19 plants. Resistant KDH13 plants systemically infected with BCTV showed only mild enation without leaf curling after 30 days. In contrast, severe leaf curling appeared after 12 days in susceptible plants with higher virus accumulation. Transcriptome analysis of the BCTV-infected KDH13 plants at the early stage of symptom development showed only 132 genes that were exclusively deregulated compared to the regulation of a large number of genes (1018 genes) in KDH19 plants. Pathway enrichment analysis showed that differentially expressed genes were predominantly involved in hormone metabolism, DNA methylation, immune response, cell cycle, biotic stress and oxidative stress. The auxin level in both resistant and susceptible plants increased in response to BCTV infection. Remarkably, exogenous application of auxin caused leaf curling phenotype in the absence of the virus. This study demonstrates the response of resistant and susceptible plants to BCTV infection at both local and systemic infections and highlights the defence-related genes and metabolic pathways including auxin for their contribution towards BCTV symptom development and resistance in sugar beet.

Genome-wide identification, phylogenetic classification of histone acetyltransferase genes, and their expression analysis in sugar beet (Beta vulgaris L.) under salt stress.

MAIN CONCLUSION: This study identified seven histone acetyltransferase-encoding genes (HATs) from Beta vulgaris L. (sugar beet) genome through bioinformatics tools and analyzed their expression profiles under salt stress. Sugar beet HATs are phylogenetically divided into four families: GNAT, MYST, CBP, and TAFII250. The BvHAT genes were differentially transcribed in leaves, stems, and roots of B. vulgaris salt-resistant (Casino) and -sensitive (Bravo) cultivars under salt stress. Histone acetylation is regulated by histone acetyltransferases (HATs), which catalyze ɛ-amino bond formation between lysine residues and acetyl groups with a cofactor, acetyl-CoA. Even though the HATs are known to participate in stress response and development in model plants, little is known about the functions of HATs in crops. In sugar beet (Beta vulgaris L.), they have not yet been identified and characterized. Here, an in silico analysis of the HAT gene family in sugar beet was performed, and their expression patterns in leaves, stems, and roots of B. vulgaris were analyzed under salt stress. Salt-resistant (Casino) and -sensitive (Bravo) beet cultivars were used for gene expression assays. Seven HATs were identified from sugar beet genome, and named BvHAG1, BvHAG2, BvHAG3, BvHAG4, BvHAC1, BvHAC2, and BvHAF1. The HAT proteins were divided into 4 groups including MYST, GNAT (GCN5, HAT1, ELP3), CBP and TAFII250. Analysis of cis-acting elements indicated that the BvHAT genes might be involved in hormonal regulation, light response, plant development, and abiotic stress response. The BvHAT genes were differentially expressed in leaves, stems, and roots under control and 300 mM NaCl. In roots of B. vulgaris cv. Bravo, the BvHAG1, BvHAG2, BvHAG4, BvHAF1, and BvHAC1 genes were dramatically expressed after 7 and 14 days of salt stress. Interestingly, the BvHAC2 gene was not expressed under both control and stress conditions. However, the expression of BvHAG2, BvHAG3, BvHAG4, BvHAC1, BvHAC2 genes showed a significant increase in response to salt stress in the roots of cv. Casino. This study provides new insights into the potential roles of histone acetyltransferases in sugar beet.

Recessive resistance against beet chlorosis virus is conferred by the eukaryotic translation initiation factor (iso)4E in Beta vulgaris.

Eukaryotic translation initiation factors (eIFs) are important for mRNA translation but also pivotal for plant-virus interaction. Most of these plant-virus interactions were found between plant eIFs and the viral protein genome-linked (VPg) of potyviruses. In case of lost interaction due to mutation or deletion of eIFs, the viral translation and subsequent replication within its host is negatively affected, resulting in a recessive resistance. Here we report the identification of the Beta vulgaris Bv-eIF(iso)4E as a susceptibility factor towards the VPg-carrying beet chlorosis virus (genus Polerovirus). Using yeast two-hybrid and bimolecular fluorescence complementation assays, the physical interaction between Bv-eIF(iso)4E and the putative BChV-VPg was detected, while the VPg of the closely related beet mild yellowing virus (BMYV) was found to interact with the two isoforms Bv-eIF4E and Bv-eIF(iso)4E. These VPg-eIF interactions within the polerovirus-beet pathosystem were demonstrated to be highly specific, as single mutations within the predicted cap-binding pocket of Bv-eIF(iso)4E resulted in a loss of interaction. To investigate the suitability of eIFs as a resistance resource against beet infecting poleroviruses, B. vulgaris plants were genome edited by CRISPR/Cas9 resulting in knockouts of different eIFs. A simultaneous knockout of the identified BMYV-interaction partners Bv-eIF4E and Bv-eIF(iso)4E was not achieved, but Bv-eIF(iso)4EKO plants showed a significantly lowered BChV accumulation and decrease in infection rate from 100% to 28.86%, while no influence on BMYV accumulation was observed. Still, these observations support that eIFs are promising candidate genes for polerovirus resistance breeding in sugar beet.

Applicability of SCoT markers in unraveling genetic variation and population structure among sugar beet (Beta vulgaris L.) germplasm.

BACKGROUND: Sugar beet (Beta vulgaris L.) holds significant importance as a crop globally cultivated for sugar production. The genetic diversity present in sugar beet accessions plays a crucial role in crop improvement programs. METHODS AND RESULTS: During the present study, we collected 96 sugar beet accessions from different regions and extracted DNA from their leaves. Genomic DNA was amplified using SCoT primers, and the resulting fragments were separated by gel electrophoresis. The data were analyzed using various genetic diversity indices, and constructed a population STRUCTURE, applied the unweighted pair-group method with arithmetic mean (UPGMA), and conducted Principle Coordinate Analysis (PCoA). The results revealed a high level of genetic diversity among the sugar beet accessions, with 265 bands produced by the 10 SCoT primers used. The percentage of polymorphic bands was 97.60%, indicating substantial genetic variation. The study uncovered significant genetic variation, leading to higher values for overall gene diversity (0.21), genetic distance (0.517), number of effective alleles (1.36), Shannon's information index (0.33), and polymorphism information contents (0.239). The analysis of molecular variance suggested a considerable amount of genetic variation, with 89% existing within the population. Using STRUCTURE and UPGMA analysis, the sugar beet germplasm was divided into two major populations. Structure analysis partitioned the germplasm based on the origin and domestication history of sugar beet, resulting in neighboring countries clustering together. CONCLUSION: The utilization of SCoT markers unveiled a noteworthy degree of genetic variation within the sugar beet germplasm in this study. These findings can be used in future breeding programs with the objective of enhancing both sugar beet yield and quality.

Effect of BvAgNP on growth, development, and glyoxalase gene expression analysis in Mammillaria bombycina and Selenicereus undatus.

BACKGROUND: Silver nanoparticles (AgNPs) have been used in plant tissue culture as growth stimulants, promoting bud initiation, germination, and rooting. In prior studies, AgNPs were synthesized and characterized by green synthesis using extracts from Beta vulgaris var. cicla (BvAgNP), and their functionality as seed disinfectant and antimicrobial was verified. In this study, we evaluated the effect of BvAgNP on the growth and development of Mammillaria bombycina and Selenicereus undatus in vitro, as well as the expression of glyoxalase genes. METHODS: Explants from M. bombycina and S. undatus in vitro were treated with 25, 50, and 100 mg/L of BvAgNP. After 90 days, morphological characteristics were evaluated, and the expression of glyoxalase genes was analyzed by qPCR. RESULTS: All treatments inhibited rooting for M. bombycina and no bud initiation was observed. S. undatus, showed a maximum response in rooting and bud generation at 25 mg/L of BvAgNP. Scanning electron microscopy (SEM) results exhibited a higher number of vacuoles in stem cells treated with BvAgNP compared to the control for both species. Expression of glyoxalase genes in M. bombycina increased in all treatments, whereas it decreased for S. undatus, however, increasing in roots. CONCLUSIONS: This study presents the effects of BvAgNP on the growth and development of M. bombycina and S. undatus, with the aim of proposing treatments that promote in vitro rooting and bud initiation.

A chromosome-level genome assembly of the beet armyworm Spodoptera exigua.

BACKGROUND: The beet armyworm Spodoptera exigua is a polyphagous caterpillar that causes serious damage to many species of crops and vegetables. To gain insight into how this polyphagous insect differs from less harmful oligophagous species, we generated a chromosome-level assembly and compared it to closely related species with the same or different feeding habits. RESULTS: Based on Illumina and Pacific Biosciences data and Hi-C technology, 425.6 Mb of genome sequences were anchored and oriented into 31 linkage groups, with an N50 length of 14.8 Mb. A total of 24,649 gene models were predicted, of which 97.4% were identified in the genome assembly. Chemosensory genes are vital for locating food: of the four main families, odorant-binding proteins, chemosensory proteins and olfactory receptors showed little difference, whereas gustatory receptors are greatly expanded in S. exigua. Examination of other polyphagous insects confirmed this difference from oligophagous congeners and further identified the bitter receptor subfamily as being particularly affected. CONCLUSION: Our high-quality genome sequence for beet armyworm identified a key expansion of the bitter gustatory receptor subfamily in this and other pests that differs crucially from more benign relatives and offers insight into the biology and possible future means of control for these economically important insects.

The Effects of DNA Methylation on Cytoplasmic Male Sterility in Sugar Beet.

DNA methylation is widely found in higher plants and can control gene expression by regulation without changing the DNA sequence. In this study, the whole-genome methylation map of sugar beet was constructed by WGBS (whole-genome bisulfite sequencing) technology, and the results of WGBS were verified by bisulfite transformation, indicating that the results of WGBS technology were reliable. In addition, 12 differential methylation genes (DMGs) were identified, which were related to carbohydrate and energy metabolism, pollen wall development, and endogenous hormone regulation. Quantitative real-time PCR (qRT-PCR) showed that 75% of DMG expression levels showed negative feedback with methylation level, indicating that DNA methylation can affect gene expression to a certain extent. In addition, we found hypermethylation inhibited gene expression, which laid a foundation for further study on the molecular mechanism of DNA methylation at the epigenetic level in sugar beet male sterility.

Genome-Wide Identification of GRAS Transcription Factors and Their Functional Analysis in Salt Stress Response in Sugar Beet.

GAI-RGA-and-SCR (GRAS) transcription factors can regulate many biological processes such as plant growth and development and stress defense, but there are few related studies in sugar beet. Salt stress can seriously affect the yield and quality of sugar beet (Beta vulgaris). Therefore, this study used bioinformatics methods to identify GRAS transcription factors in sugar beet and analyzed their structural characteristics, evolutionary relationships, regulatory networks and salt stress response patterns. A total of 28 BvGRAS genes were identified in the whole genome of sugar beet, and the sequence composition was relatively conservative. According to the topology of the phylogenetic tree, BvGRAS can be divided into nine subfamilies: LISCL, SHR, PAT1, SCR, SCL3, LAS, SCL4/7, HAM and DELLA. Synteny analysis showed that there were two pairs of fragment replication genes in the BvGRAS gene, indicating that gene replication was not the main source of BvGRAS family members. Regulatory network analysis showed that BvGRAS could participate in the regulation of protein interaction, material transport, redox balance, ion homeostasis, osmotic substance accumulation and plant morphological structure to affect the tolerance of sugar beet to salt stress. Under salt stress, BvGRAS and its target genes showed an up-regulated expression trend. Among them, BvGRAS-15, BvGRAS-19, BvGRAS-20, BvGRAS-21, LOC104892636 and LOC104893770 may be the key genes for sugar beet's salt stress response. In this study, the structural characteristics and biological functions of BvGRAS transcription factors were analyzed, which provided data for the further study of the molecular mechanisms of salt stress and molecular breeding of sugar beet.

MicroRNAs Participate in Morphological Acclimation of Sugar Beet Roots to Nitrogen Deficiency.

Nitrogen (N) is essential for sugar beet (Beta vulgaris L.), a highly N-demanding sugar crop. This study investigated the morphological, subcellular, and microRNA-regulated responses of sugar beet roots to low N (LN) stress (0.5 mmol/L N) to better understand the N perception, uptake, and utilization in this species. The results showed that LN led to decreased dry weight of roots, N accumulation, and N dry matter production efficiency, along with damage to cell walls and membranes and a reduction in organelle numbers (particularly mitochondria). Meanwhile, there was an increase in root length (7.2%) and branch numbers (29.2%) and a decrease in root surface area (6.14%) and root volume (6.23%) in sugar beet after 7 d of LN exposure compared to the control (5 mmol/L N). Transcriptomics analysis was confirmed by qRT-PCR for 6 randomly selected microRNAs, and we identified 22 differentially expressed microRNAs (DEMs) in beet root under LN treatment. They were primarily enriched in functions related to binding (1125), ion binding (641), intracellular (437) and intracellular parts (428), and organelles (350) and associated with starch and sucrose metabolism, tyrosine metabolism, pyrimidine metabolism, amino sugar and nucleotide sugar metabolism, and isoquinoline alkaloid biosynthesis, as indicated by the GO and KEGG analyses. Among them, the upregulated miR156a, with conserved sequences, was identified as a key DEM that potentially targets and regulates squamosa promoter-binding-like proteins (SPLs, 104889216 and 104897537) through the microRNA-mRNA network. Overexpression of miR156a (MIR) promoted root growth in transgenic Arabidopsis, increasing the length, surface area, and volume. In contrast, silencing miR156a (STTM) had the opposite effect. Notably, the fresh root weight decreased by 45.6% in STTM lines, while it increased by 27.4% in MIR lines, compared to the wild type (WT). It can be inferred that microRNAs, especially miR156, play crucial roles in sugar beet root's development and acclimation to LN conditions. They likely facilitate active responses to N deficiency through network regulation, enabling beet roots to take up nutrients from the environment and sustain their vital life processes.

Expression and Functional Identification of SPL6/7/9 Genes under Drought Stress in Sugarbeet Seedlings.

Sugar beet is a significant sugar crop in China, primarily cultivated in arid regions of the north. However, drought often affects sugar beet cultivation, leading to reduced yield and quality. Therefore, understanding the impact of drought on sugar beets and studying their drought tolerance is crucial. Previous research has examined the role of SPL (SQUAMOSA promoter-binding protein-like) transcription factors in plant stress response; however, the precise contribution of SPLs to the drought stress response in sugar beets has yet to be elucidated. In this study, we identified and examined the BvSPL6, BvSPL7, and BvSPL9 genes in sugar beets, investigating their performance during the seedling stage under drought stress. We explored their drought resistance characteristics using bioinformatics, quantitative analysis, physiological experiments, and molecular biology experiments. Drought stress and rehydration treatments were applied to sugar beet seedlings, and the expression levels of BvSPL6, BvSPL7, and BvSPL9 genes in leaves were quantitatively analyzed at 11 different time points to evaluate sugar beets' response and tolerance to drought stress. Results indicated that the expression level of the BvSPL6/9 genes in leaves was upregulated during the mid-stage of drought stress and downregulated during the early and late stages. Additionally, the expression level of the BvSPL7 gene gradually increased with the duration of drought stress. Through analyzing changes in physiological indicators during different time periods of drought stress and rehydration treatment, we speculated that the regulation of BvSPL6/7/9 genes is associated with sugar beet drought resistance and their participation in drought stress response. Furthermore, we cloned the CDS sequences of BvSPL6, BvSPL7, and BvSPL9 genes from sugar beets and conducted sequence alignment with the database to validate the results. Subsequently, we constructed overexpression vectors, named 35S::BvSPL6, 35S::BvSPL7, and 35S::BvSPL9, and introduced them into sugar beets using Agrobacterium-mediated methods. Real-time fluorescence quantitative analysis revealed that the expression levels of BvSPL6/7/9 genes in transgenic sugar beets increased by 40% to 80%. The drought resistance of transgenic sugar beets was significantly enhanced compared with the control group.

Genomic characterization of a nematode tolerance locus in sugar beet.

BACKGROUND: Infection by beet cyst nematodes (BCN, Heterodera schachtii) causes a serious disease of sugar beet, and climatic change is expected to improve the conditions for BCN infection. Yield and yield stability under adverse conditions are among the main breeding objectives. Breeding of BCN tolerant sugar beet cultivars offering high yield in the presence of the pathogen is therefore of high relevance. RESULTS: To identify causal genes providing tolerance against BCN infection, we combined several experimental and bioinformatic approaches. Relevant genomic regions were detected through mapping-by-sequencing using a segregating F2 population. DNA sequencing of contrasting F2 pools and analyses of allele frequencies for variant positions identified a single genomic region which confers nematode tolerance. The genomic interval was confirmed and narrowed down by genotyping with newly developed molecular markers. To pinpoint the causal genes within the potential nematode tolerance locus, we generated long read-based genome sequence assemblies of the tolerant parental breeding line Strube U2Bv and the susceptible reference line 2320Bv. We analyzed continuous sequences of the potential locus with regard to functional gene annotation and differential gene expression upon BCN infection. A cluster of genes with similarity to the Arabidopsis thaliana gene encoding nodule inception protein-like protein 7 (NLP7) was identified. Gene expression analyses confirmed transcriptional activity and revealed clear differences between susceptible and tolerant genotypes. CONCLUSIONS: Our findings provide new insights into the genomic basis of plant-nematode interactions that can be used to design and accelerate novel management strategies against BCN.

CRISPR/Cas9-mediated methoprene-tolerant 1 knockout results in precocious metamorphosis of beet armyworm (Spodoptera exigua) only at the late larval stage.

Juvenile hormone (JH) controls almost every aspect of an insect, especially metamorphosis. Since RNA interference works on transcripts and is often insufficient in Lepidoptera, how JH affects larval development in these insects is not well studied. Using the CRISPR/Cas9 technique, we knocked out Spodoptera exigua methoprene-tolerant 1 (SeMet1) gene of beet armyworm by modifying two sites in the coding region. However, SeMet1 knockout did not affect egg hatch rate or larval development at L1-L3 stages. In contrast to the consistent five larval instars of the control group, L4 SeMet1 mutants began to show signs of precocious metamorphosis, that is, small patches of pupal cuticle. Most L4 and all L5 SeMet1 mutants died for failing to shed their mosaic cuticles. RNA-seq indicated that most genes encoding pupal cuticle proteins and chitinase genes were altered in SeMet1 mutant L4 larvae. SeKr-h1, a key transcription factor in JH action was significantly down-regulated in L3-L5 larvae, while SeBR-C, a pupal indicator was only upregulated in L4-L5 larvae. These results suggested that S. exigua larvae may initially develop independently of JH, and involve SeMet1 in transducing JH signalling, leading to controlled larval metamorphosis at the late larval stage. We believe our findings will enhance better understanding of JH regulation of larval development.

Assessment of co-infection with BNYVV and BSCTV on resistance against Rhizomania disease in transgenic sugar beet plants.

Sugar beet is an economically important crop and one of the major sources of sucrose around the world. Beet necrotic yellow vein virus (BNYVV) and Beet severe curly top virus (BSCTV) are two widespread viruses in sugar beet that cause severe damage to its performance. Previously, we have successfully produced resistance to BNYVV based on RNA silencing in sugar beet by introducing constructs carrying the viral coat-protein-encoding DNA sequence, CP21, in sense and anti-sense orientations. Yet, the RNA silencing-mediated resistance to a specific virus could be affected by other ones as a part of synergistic interactions. In this study, we assayed the specificity of the induced resistance against BNYVV in two sets of transgenic events, S3 and S6 carrying 5'-UTR with or without CP21-coding sequences, respectively. These events were subjected to viral challenges with either BNYVV, an Iranian isolate of BSCTV (BSCTV-Ir) or both. All the plants inoculated with just BSCTV-Ir displayed curly-leaf symptoms. However, partial resistance was evident in S3 events as shown by mild symptoms and reduced PCR amplification of the BSCTV-Ir coat protein encoding sequence. Based on the presented data, resistance to BNYVV was stable in almost all the transgenic plants co-infected with BSCTV-Ir, except for one event, S3-229. In general, it seems that the co-infection does not affect the resistance to BNYVV in transgenic plants. These findings demonstrated that the introduced RNA silencing-mediated resistance against BNYVV in transgenic sugar beets is specific and is not suppressed after co-infection with a heterologous virus.

Effects of cadmium stress on the morphology, physiology, cellular ultrastructure, and BvHIPP24 gene expression of sugar beet (Beta vulgaris L.).

To clarify the mechanism of the response of sugar beet (Beta vulgaris L.) to cadmium (Cd) stress, this study investigated changes in the phenotype, physiological indexes, and subcellular structure of B. vulgaris under Cd treatment and the transcriptional pattern of the BvHIPP24 gene (a heavy metal-associated isoprenylated plant protein involved in heavy metal detoxification). The plant height and shoot and root growth of B. vulgaris seedlings were inhibited to some extent under 0.5 and 1 mM Cd, with gradually wilting and yellowing of leaves and dark brown roots. When the Cd concentration was increased, malondialdehyde content and the activities of peroxidase, superoxide dismutase, and glutathione S-transferase increased differentially. qPCR indicated that the expression of BvHIPP24 was induced by different concentrations of Cd. Although transmission electron microscopy revealed damage to nuclei, mitochondria, and chloroplasts, B. vulgaris exhibited strong adaptability to 0.5 mM Cd according to a comprehensive analysis using the membership function. The results showed that B. vulgaris may reduce cell damage and improve its Cd tolerance by regulating functional gene expression and antioxidant enzymes. This study increases our understanding of the Cd-tolerance mechanism of B. vulgaris and provides insights into the use of B. vulgaris in Cd bioremediation.

Sugar beet is a novel energy crop with superior characteristics for both heavy metal phytoremediation and biomass energy development. This work is the first to investigate both the morphological, physiological, and ultrastructural response of sugar beet to cadmium stress and the induction of a functional metallochaperone gene by cadmium. This study explains the cadmium tolerance mechanism of sugar beet based on a comprehensive evaluation and provides an important theoretical basis for further application of beet in heavy metal bioremediation.

Combined Omics Approaches Reveal Distinct Mechanisms of Resistance and/or Susceptibility in Sugar Beet Double Haploid Genotypes at Early Stages of Beet Curly Top Virus Infection.

Sugar beet is susceptible to Beet curly top virus (BCTV), which significantly reduces yield and sugar production in the semi-arid growing regions worldwide. Sources of genetic resistance to BCTV is limited and control depends upon insecticide seed treatments with neonicotinoids. Through double haploid production and genetic selection, BCTV resistant breeding lines have been developed. Using BCTV resistant (R) [KDH13; Line 13 and KDH4-9; Line 4] and susceptible (S) [KDH19-17; Line 19] lines, beet leafhopper mediated natural infection, mRNA/sRNA sequencing, and metabolite analyses, potential mechanisms of resistance against the virus and vector were identified. At early infection stages (2- and 6-days post inoculation), examples of differentially expressed genes highly up-regulated in the 'R' lines (vs. 'S') included EL10Ac5g10437 (inhibitor of trypsin and hageman factor), EL10Ac6g14635 (jasmonate-induced protein), EL10Ac3g06016 (ribosome related), EL10Ac2g02812 (probable prolyl 4-hydroxylase 10), etc. Pathway enrichment analysis showed differentially expressed genes were predominantly involved with peroxisome, amino acids metabolism, fatty acid degradation, amino/nucleotide sugar metabolism, etc. Metabolite analysis revealed significantly higher amounts of specific isoflavonoid O-glycosides, flavonoid 8-C glycosides, triterpenoid, and iridoid-O-glycosides in the leaves of the 'R' lines (vs. 'S'). These data suggest that a combination of transcriptional regulation and production of putative antiviral metabolites might contribute to BCTV resistance. In addition, genome divergence among BCTV strains differentially affects the production of small non-coding RNAs (sncRNAs) and small peptides which may potentially affect pathogenicity and disease symptom development.