ABSTRACT
Polyploidization leads to novel phenotypes and is a major force in evolution. However, the relationship between the evolution of new traits and variations in the post-translational modifications (PTM) of proteins during polyploidization has not been studied. Acetylation of lysine residues is a common protein PTM that plays a critical regulatory role in central metabolism. To test whether changes in metabolism in citrus fruit is associated with the reprogramming of lysine acetylation (Kac) in non-histone proteins during allotetraploidization, we performed a global acetylome analysis of fruits from a synthetic allotetraploid citrus and its diploid parents. A total of 4,175 Kac sites were identified on 1,640 proteins involved in a wide range of fruit traits. In the allotetraploid, parental dominance (i.e. resemblance to one of the two parents) in specific fruit traits, such as fruit acidity and flavonol metabolism, was highly associated with parental Kac level dominance in pertinent enzymes. This association is due to Kac-mediated regulation of enzyme activity. Moreover, protein Kac probably contributes to the discordance between the transcriptomic and proteomic variations during allotetraploidization. The acetylome reprogramming can be partially explained by the expression pattern of several lysine deacetylases (KDACs). Overexpression of silent information regulator 2 (CgSRT2) and histone deacetylase 8 (CgHDA8) diverted metabolic flux from primary metabolism to secondary metabolism and partially restored a metabolic status to the allotetraploid, which expressed attenuated levels of CgSRT2 and CgHDA8. Additionally, KDAC inhibitor treatment greatly altered metabolism in citrus fruit. Collectively, these findings reveal the important role of acetylome reprogramming in trait evolution during polyploidization.
Subject(s)
Citrus , Proteomics , Lysine/metabolism , Proteome/genetics , Proteome/metabolism , Fruit/metabolism , Citrus/genetics , Citrus/metabolism , Acetylation , Protein Processing, Post-TranslationalABSTRACT
KEY MESSAGE: Overexpression of the salicylic acid binding protein 2 (SABP2) gene from Tobacco results in enhanced tolerance to Huanglongbing (HLB; citrus greening disease) in transgenic sweet oranges. Huanglongbing (HLB), the most destructive citrus disease, is caused by Candidatus Liberibacter asiaticus (CaLas). Currently, no cure for this disease exists, and all commercially planted cultivars are highly susceptible. Salicylic Acid Binding Protein 2 (SABP2) is a well-characterized protein essential for establishing systemic acquired resistance (SAR) in tobacco. The constitutive over expression of SABP2 from tobacco (NtSABP2) in 'Hamlin' sweet orange resulted in the production of several transgenic lines with variable transcript levels. Transient expression of the NtSABP2-EGFP fusion protein in Nicotiana benthamiana plants demonstrated that NtSABP2 was cytosolic in its subcellular localization. In a long-term field study, we identified a SABP2 transgenic line with significantly reduced HLB symptoms that maintained a consistently low CaLas titer. Transcriptome analysis of this selected transgenic line demonstrated upregulation of several genes related to plant defense and SAR pathways. Genes, such as NPR family genes and those coding for monooxygenases and lipoxygenases, were upregulated in the 35S-NtSABP2 overexpressing line and might be candidates for incorporation into our citrus improvement program.
Subject(s)
Citrus sinensis , Citrus , Rhizobiaceae , Nicotiana/genetics , Citrus/genetics , Plant Diseases/genetics , Citrus sinensis/genetics , Citrus sinensis/metabolism , Liberibacter , Salicylic Acid/metabolismABSTRACT
BACKGROUND: Development of precise genome editing strategies is a prerequisite for producing edited plants that can aid in the study of gene function and help understand the genetic traits in a cultivar. Citrus embryogenic cell cultures can be used to rapidly produce a large population of genome edited transformed citrus lines. The ability to introduce specific mutations in the genome of these cells using two constructs (pC-PDS1 and pC-PDS2) was evaluated in this study. RESULTS: Citrus sinensis 'EV2' embryogenic cell cultures are amenable to Agrobacterium-mediated CRISPR/Cas9-based genome editing. Guide RNAs (gRNAs) targeting two locations in the phytoene desaturase (PDS) gene were either driven by the Arabidopsis U6-26 promoter (pC-PDS1) or assembled as a Csy4 array under the control of the CmYLCV promoter (pC-PDS2). All transgenic embryos were completely albino and no variegated phenotype was observed. We evaluated 12 lines from each construct in this study and the majority contain either insertion (1-2 bp), substitution (1 bp), or deletion (1-3 bp) mutations that occurred close to the protospacer adjacent motif. CONCLUSIONS: Both the pC-PDS1 and pC-PDS2 could successfully edit the citrus embryogenic cell cultures. However, the editing efficiency was dependent on the gRNA, confirming that the selection of a proper gRNA is essential for successful genome editing using the CRISPR/Cas9 technique. Also, utilization of embryogenic cell cultures offers another option for successful genome editing in citrus.
Subject(s)
CRISPR-Cas Systems , Cell Culture Techniques/methods , Citrus/genetics , Gene Editing/methods , Agrobacterium/genetics , Base Sequence , Citrus/embryology , Clustered Regularly Interspaced Short Palindromic Repeats , Genome, Plant , Mutation , Oxidoreductases/genetics , Plants, Genetically Modified/genetics , RNA, Guide, Kinetoplastida/geneticsABSTRACT
KEY MESSAGE: The physical locations of citrus centromere are revealed by combining genetic and immunological assays for the first time and nine citrus centromere-specific markers for cytogenetics are mined. Centromere localization is challenging, because highly redundant repetitive sequences in centromeric regions make sequence assembly difficult. Although several citrus genomes have been released, the centromeric regions and their characteristics remain to be elucidated. Here, we mapped citrus centromeres through half-tetrad analysis (HTA) that included the genotyping of 54 tetraploid hybrids derived from 2n megagametophytes of Nadorcott tangor with 212 single nucleotide polymorphism (SNP) markers. The sizes of centromeric regions, which estimated based on the heterozygosity restitution rate pattern along the chromosomes, ranged from 1.12 to 18.19 Mb. We also profiled the binding sequences with the centromere-specific histone variant CenH3 by chromatin immunoprecipitation sequencing (ChIP-seq). Based on the positions of the top ten CenH3-enriched contigs, the sizes of centromeric regions were estimated to range from 0.01 to 7.60 Mb and were either adjacent to or included in the centromeric regions identified by HTA. We used DNA probes from two repeats selected from the centromeric regions and seven CenH3-binding centromeric repeats to verify centromeric locations by fluorescence in situ hybridization (FISH). Centromere localization in citrus will contribute to the mining of centromeric/pericentromeric markers, thus to facilitate the rapid identification of mechanisms underlying 2n gamete formation and serve the polyploidy breeding.
Subject(s)
Centromere/genetics , Citrus/genetics , Cytogenetics/methods , Antibody Specificity , Chromatin Immunoprecipitation Sequencing , Genes, Plant/immunology , Genotyping Techniques/methods , In Situ Hybridization, Fluorescence , Polymorphism, Single Nucleotide , TetraploidyABSTRACT
Huanglongbing (HLB), a bacterial disease caused by Candidatus Liberibacter asiaticus (CLas), is a major threat to the citrus industry. In a previous study conducted by our laboratory, several citrus transgenic trees expressing the Arabidopsis thaliana NPR1 (AtNPR1) gene remained HLB-free when grown in a field site under high HLB disease pressure. To determine the molecular mechanisms behind AtNPR1-mediated tolerance to HLB, a transcriptome analysis was performed using AtNPR1 overexpressing transgenic trees and non-transgenic trees as control, from which we identified 57 differentially expressed genes (DEGs). Data mining revealed the enhanced transcription of genes encoding pathogen-associated molecular patterns (PAMPs), transcription factors, leucine-rich repeat receptor kinases (LRR-RKs), and putative ankyrin repeat-containing proteins. These proteins were highly upregulated in the AtNPR1 transgenic line compared to the control plant. Furthermore, analysis of protein-protein interactions indicated that AtNPR1 interacts with CsNPR3 and CsTGA5 in the nucleus. Our results suggest that AtNPR1 positively regulates the innate defense mechanisms in citrus thereby boosting resistance and effectively protecting the plant against HLB.
Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Citrus sinensis/genetics , Disease Resistance/genetics , Disease Resistance/physiology , Basic-Leucine Zipper Transcription Factors/genetics , Citrus sinensis/immunology , Citrus sinensis/microbiology , Gene Expression Profiling , Gene Expression Regulation, Plant , Genes, Plant/genetics , Immunity, Innate , Liberibacter , Plant Diseases/immunology , Plant Diseases/microbiology , Plant Infertility/physiology , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/immunology , Transcription Factors/metabolism , TranscriptomeABSTRACT
Genetic engineering approaches offer an alternative method to the conventional breeding of Citrus sp. 'W. Murcott' mandarin (a hybrid of 'Murcott' and an unknown pollen parent) is one of the most commercially important cultivars grown in many regions around the world. Transformation of 'W. Murcott' mandarin was achieved by direct DNA uptake using a protoplast transformation system. DNA construct (pAO3), encoding Green Fluorescent Protein (GFP) and the cDNA of Xa21, a Xanthomonas resistance gene from rice, was used to transform protoplasts of 'W. Murcott' mandarin. Following citrus protoplast culture and regeneration, transformed micro calli were microscopically designated via GFP expression, physically isolated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. More than 150 transgenic embryos were recovered and from them, ten transgenic lines were regenerated and cultured on rooting medium for shoot elongation. Transgenic shoots were micrografted and established in the greenhouse with 3-5 replicates per line. The insertion of Xa21 and GFP was confirmed by PCR and southern blot analysis. GFP expression was verified by fluorescence microscopy and western blot analysis revealed expression of Xa21 although it was variable among transgenic lines, as shown by RT-qPCR. Transgenic plants challenged with the citrus canker pathogen by syringe inoculation showed a reduction in lesion number and bacterial populations within lesions compared to non-transgenic control plants. Transgenic 'W. Murcott' mandarin lines with improved canker resistance via protoplast transformation from embryogenic callus with the Xa21 gene from rice are being evaluated under field conditions to validate the level of resistance.
Subject(s)
Citrus/genetics , Oryza/genetics , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Protein Serine-Threonine Kinases/genetics , Citrus/growth & development , Citrus/microbiology , Disease Resistance/genetics , Oryza/microbiology , Plant Diseases/genetics , Plant Diseases/microbiology , Plant Proteins/administration & dosage , Plants, Genetically Modified/drug effects , Plants, Genetically Modified/microbiology , Protein Serine-Threonine Kinases/administration & dosage , Protoplasts/microbiologyABSTRACT
BACKGROUND: Citrus blight is a citrus tree overall decline disease and causes serious losses in the citrus industry worldwide. Although it was described more than one hundred years ago, its causal agent remains unknown and its pathophysiology is not well determined, which hampers our understanding of the disease and design of suitable disease management. RESULTS: In this study, we sequenced and assembled the draft genome for Swingle citrumelo, one important citrus rootstock. The draft genome is approximately 280 Mb, which covers 74 % of the estimated Swingle citrumelo genome and the average coverage is around 15X. The draft genome of Swingle citrumelo enabled us to conduct transcriptome analysis of roots of blight and healthy Swingle citrumelo using RNA-seq. The RNA-seq was reliable as evidenced by the high consistence of RNA-seq analysis and quantitative reverse transcription PCR results (R(2) = 0.966). Comparison of the gene expression profiles between blight and healthy root samples revealed the molecular mechanism underneath the characteristic blight phenotypes including decline, starch accumulation, and drought stress. The JA and ET biosynthesis and signaling pathways showed decreased transcript abundance, whereas SA-mediated defense-related genes showed increased transcript abundance in blight trees, suggesting unclassified biotrophic pathogen was involved in this disease. CONCLUSIONS: Overall, the Swingle citrumelo draft genome generated in this study will advance our understanding of plant biology and contribute to the citrus breeding. Transcriptome analysis of blight and healthy trees deepened our understanding of the pathophysiology of citrus blight.
Subject(s)
Gene Expression Profiling , Genome, Plant , Plant Diseases/genetics , Plant Roots/genetics , Transcriptome , Computational Biology/methods , Disease Resistance/genetics , Gene Ontology , Genomics/methods , High-Throughput Nucleotide Sequencing , Metabolic Networks and Pathways , Molecular Sequence Annotation , Plant Roots/metabolismABSTRACT
Comprehensive and quantitative proteomic information on citrus floral bud is significant for understanding male sterility of the cybrid pummelo (G1+HBP) with nuclear genome of HBP and foreign mitochondrial genome of G1. Scanning electron microscopy and transmission electron microscopy analyses of the anthers showed that the development of pollen wall in G1+HBP was severely defective with a lack of exine and sporopollenin formation. Proteomic analysis was used to identify the differentially expressed proteins between male sterile G1+HBP and fertile type (HBP) with the aim to clarify their potential roles in anther development and male sterility. On the basis of iTRAQ quantitative proteomics, we identified 2235 high-confidence protein groups, 666 of which showed differentially expressed profiles in one or more stages. Proteins up- or down-regulated in G1+HBP were mainly involved in carbohydrate and energy metabolism (e.g., pyruvate dehydrogenase, isocitrate dehydrogenase, ATP synthase, and malate dehydrogenase), nucleotide binding (RNA-binding proteins), protein synthesis and degradation (e.g., ribosome proteins and proteasome subunits). Additionally, the proteins located in mitochondria also showed changed expression patterns. These findings provide a valuable inventory of proteins involved in floral bud development and contribute to elucidate the mechanism of cytoplasmic male sterility in the cybrid pummelo.
Subject(s)
Citrus/metabolism , Flowers/metabolism , Mitochondrial Proteins/metabolism , Plant Proteins/metabolism , Proteome/metabolism , Carbohydrate Metabolism , Citrus/cytology , Citrus/growth & development , Flowers/cytology , Flowers/growth & development , Gene Expression Regulation, Plant , Mitochondrial Proteins/genetics , Molecular Sequence Annotation , Plant Proteins/genetics , Proteomics , Reproduction , Staining and Labeling , TranscriptomeABSTRACT
KEY MESSAGE: 2 n megagametophyte formation plays an important role in polyploidization in polyembryonic citrus and is valuable for plant improvement. Tetraploid plants are frequently observed in the seedlings of diploid polyembryonic citrus genotypes. However, the mechanisms underlying the formation of tetraploids are still indistinct when apomictic citrus genotypes are used as female parent to cross with tetraploids. Herein, 54 tetraploid progenies, which were unexpectedly obtained previously from four 2x × 4x crosses using polyembryonic 'Nadorcott' tangor as seed parent, were analyzed by 22 simple sequence repeat (SSR) markers, aiming to reveal their genetic origin and the mechanism underlying 2n megagametophyte formation. The results showed that 13 tetraploids from all these four crosses were doubled diploids as indicated by their identical SSR allelic profile with their female parent; while the remaining 41 tetraploids apparently exhibited paternally derived alleles, which confirmed their zygotic origin. Furthermore, the genotyping of all hybrids indicated that all of them arose from 2n megagametophytes. Based on the genotypes of 2n megagametophytes, the analysis of maternal heterozygosity restitution (HR) for each marker showed that it varied from 0.00 to 87.80 % with a mean value of 40.89 %. In addition, it was observed that 13 markers displayed a lower rate than 50 %. On the basis of the above results, it can be speculated that the second division restitution (SDR) is the mechanism underlying the 2n megagametophyte formation in 'Nadorcott' tangor. The elucidation of the mechanism of 2n megagametophyte formation will be of great help to optimize further sexual hybridization for polyploids in citrus.
Subject(s)
Citrus/genetics , Tetraploidy , Diploidy , Genotype , Seedlings/geneticsABSTRACT
Introduction: Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a devastating disease worldwide. Previously, we successfully generated canker-resistant Citrus sinensis cv. Hamlin lines in the T0 generation. This was achieved through the transformation of embryogenic protoplasts using the ribonucleoprotein (RNP) containing Cas12a and one crRNA to edit the canker susceptibility gene, CsLOB1, which led to small indels. Methods: Here, we transformed embryogenic protoplasts of Hamlin with RNP containing Cas12a and three crRNAs. Results: Among the 10 transgene-free genome-edited lines, long deletions were obtained in five lines. Additionally, inversions were observed in three of the five edited lines with long deletions, but not in any edited lines with short indel mutations, suggesting long deletions maybe required for inversions. Biallelic mutations were observed for each of the three target sites in four of the 10 edited lines when three crRNAs were used, demonstrating that transformation of embryogenic citrus protoplasts with Cas12a and three crRNAs RNP can be very efficient for multiplex editing. Our analysis revealed the absence of off-target mutations in the edited lines. These cslob1 mutant lines were canker- resistant and no canker symptoms were observed after inoculation with Xcc and Xcc growth was significantly reduced in the cslob1 mutant lines compared to the wild type plants. Discussion: Taken together, RNP (Cas12a and three crRNAs) transformation of embryogenic protoplasts of citrus provides a promising solution for transgene-free multiplex genome editing with high efficiency and for deletion of long fragments.
ABSTRACT
Citrus canker disease affects citrus production. This disease is caused by Xanthomonas citri subsp. citri (Xcc). Previous studies confirmed that during Xcc infection, PthA4, a transcriptional activator like effector (TALE), is translocated from the pathogen to host plant cells. PthA4 binds to the effector binding elements (EBEs) in the promoter region of canker susceptibility gene LOB1 (EBEPthA4-LOBP) to activate its expression and subsequently cause canker symptoms. Previously, the Cas12a/CBE co-editing method was employed to disrupt EBEPthA4-LOBP of pummelo, which is highly homozygous. However, most commercial citrus cultivars are heterozygous hybrids and more difficult to generate homozygous/biallelic mutants. Here, we employed Cas12a/CBE co-editing method to edit EBEPthA4-LOBP of Hamlin (Citrus sinensis), a commercial heterozygous hybrid citrus cultivar grown worldwide. Binary vector GFP-p1380N-ttLbCas12a:LOBP1-mPBE:ALS2:ALS1 was constructed and shown to be functional via Xcc-facilitated agroinfiltration in Hamlin leaves. This construct allows the selection of transgene-free regenerants via GFP, edits ALS to generate chlorsulfuron-resistant regenerants as a selection marker for genome editing resulting from transient expression of the T-DNA via nCas9-mPBE:ALS2:ALS1, and edits gene(s) of interest (i.e., EBEPthA4-LOBP in this study) through ttLbCas12a, thus creating transgene-free citrus. Totally, 77 plantlets were produced. Among them, 8 plantlets were transgenic plants (#HamGFP1 - #HamGFP8), 4 plantlets were transgene-free (#HamNoGFP1 - #HamNoGFP4), and the rest were wild type. Among 4 transgene-free plantlets, three lines (#HamNoGFP1, #HamNoGFP2 and #HamNoGFP3) contained biallelic mutations in EBEpthA4, and one line (#HamNoGFP4) had homozygous mutations in EBEpthA4. We achieved 5.2% transgene-free homozygous/biallelic mutation efficiency for EBEPthA4-LOBP in C. sinensis cv. Hamlin, compared to 1.9% mutation efficiency for pummelo in a previous study. Importantly, the four transgene-free plantlets and 3 transgenic plantlets that survived were resistant against citrus canker. Taken together, Cas12a/CBE co-editing method has been successfully used to generate transgene-free canker-resistant C. sinensis cv. Hamlin in the T0 generation via biallelic/homozygous editing of EBEpthA4 of the canker susceptibility gene LOB1.
ABSTRACT
Huanglongbing (HLB) is one of the most destructive citrus diseases, mainly caused by the Gram-negative bacteria Candidatus Liberibacter asiaticus. Aiming at unraveling the mechanisms of different scion/rootstock combinations on improving HLB-affected orange juice quality, the effects of rootstocks on the metabolites of HLB-affected sweet orange juices were investigated using a combined strategy of untargeted metabolomics and machine learning. A total of 2531 ion features were detected using UHPLC-Q-Orbitrap high-resolution electrospray ionization mass spectrometry, and 54 metabolites including amino acids, amines, flavonoids, coumarins, fatty acids, and glycosides were definitely or tentatively identified as the differential markers based on the random forest algorithm. Furthermore, 24 metabolites were verified and semi-quantified using authentic standards. Notably, the presence of specific amino acids and amines, especially polyamines, indicated that different rootstocks might affect glutamate, aspartate, proline, and arginine metabolism to regulate the physiological response against HLB. Meanwhile, the production of flavonoids and prenylated coumarins suggested that rootstocks influenced phenylalanine and phenylpropanoid metabolism. The possible metabolic pathways were proposed, and the important intermediates were verified by authentic standards. These results provide new insights on the effects of rootstocks on the metabolites of HLB-affected sweet orange juices.
Subject(s)
Citrus sinensis , Citrus , Rhizobiaceae , Citrus sinensis/chemistry , Rhizobiaceae/physiology , Citrus/microbiology , Amino Acids/metabolism , Amines , Flavonoids/metabolism , Machine Learning , Plant Diseases/microbiologyABSTRACT
Citrus greening disease or Huanglongbing (HLB) is one of the most destructive diseases affecting all varieties of citrus worldwide. Aimed at optimizing the scion/rootstock combination to improve HLB-affected orange juice quality, a flavoromics strategy was used to investigate the effects of six different rootstocks (CH, blue, 1804, FG, SW, and Volk) on flavor quality of HLB affected orange juices. A sensory quality test was conducted by a panel to evaluate the sensory attributes of different orange juices. The orange juice from rootstock CH had the best flavor quality with highest sweetness, low sourness and bitterness, while rootstocks Volk and FG produced the poorest quality orange juices. Chemical profile analysis resulted in semi-quantification of 89 metabolites including 57 nonvolatile compounds and 32 volatile compounds using UHPLC-MS and GC-MS, respectively. Canonical correlation analysis indicated that some specific sugar and sugar alcohols including raffinose, xylose, rhamnose, glucose, sorbitol, and myo-inositol made a strong positive contribution to sweetness. Meanwhile, several amino acids including alanine, glutamic acid, proline, arginine, serine, asparagine, as well as aspartic acid were responsible for positive flavor quality. On the other hand, some nucleotides and limonin increased bitterness. In addition, KEGG pathway enrichment analysis demonstrated different rootstocks could affect aminoacyl-tRNA biosynthesis, ABC transporters, and monoterpenoid biosynthesis. These results indicated different rootstocks can change specific metabolites and thus affect the flavor quality of orange juices. This study also provides reference for optimizing the scion/rootstock combination to improve HLB-affected orange juice quality.
ABSTRACT
Citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is a destructive citrus disease worldwide. Generating disease-resistant cultivars is the most effective, environmentally friendly and economic approach for disease control. However, citrus traditional breeding is lengthy and laborious. Here, we develop transgene-free canker-resistant Citrus sinensis lines in the T0 generation within 10 months through transformation of embryogenic protoplasts with Cas12a/crRNA ribonucleoprotein to edit the canker susceptibility gene CsLOB1. Among the 39 regenerated lines, 38 are biallelic/homozygous mutants, demonstrating a 97.4% biallelic/homozygous mutation rate. No off-target mutations are detected in the edited lines. Canker resistance of the cslob1-edited lines results from both abolishing canker symptoms and inhibiting Xcc growth. The transgene-free canker-resistant C. sinensis lines have received regulatory approval by USDA APHIS and are exempted from EPA regulation. This study provides a sustainable and efficient citrus canker control solution and presents an efficient transgene-free genome-editing strategy for citrus and other crops.
Subject(s)
Citrus sinensis , Citrus , Xanthomonas , Citrus sinensis/genetics , RNA, Guide, CRISPR-Cas Systems , CRISPR-Cas Systems/genetics , Disease Resistance/genetics , Plant Breeding , Citrus/genetics , Xanthomonas/genetics , Plant Diseases/geneticsABSTRACT
Plant genomes are comprised of nuclear, plastid and mitochondrial components characterized by different patterns of inheritance and evolution. Genetic markers from the three genomes provide complementary tools for investigations of inheritance, genetic relationships and phenotypic contributions. Plant mitochondrial genomes are challenging for universal marker development because they are highly variable in terms of size, gene order and intergenic sequences and highly conserved with respect to protein-coding sequences. PCR amplification of introns with primers that anneal to conserved, flanking exons is effective for the development of polymorphic nuclear genome markers. The potential for plant mitochondrial intron polymorphisms to distinguish between congeneric species or intraspecific varieties has not been systematically investigated and is possibly constrained by requirements for intron secondary structure and interactions with co-evolved organelle intron splicing factors. To explore the potential for broadly applicable plant mitochondrial intron markers, PCR primer sets based upon conserved sequences flanking 11 introns common to seven angiosperm species were tested across a range of plant orders. PCR-amplified introns were screened for indel polymorphisms among a group of cross-compatible Citrus species and relatives; two Raphanus sativus mitotypes; representatives of the two Phaseolus vulgaris gene pools; and congeneric pairs of Cynodon, Cenchrus, Solanum, and Vaccinium species. All introns were successfully amplified from each plant entry. Length polymorphisms distinguishable by gel electrophoresis were common among genera but infrequent within genera. Sequencing of three introns amplified from 16 entries identified additional short indel polymorphisms and nucleotide substitutions that separated Citrus, Cynodon, Cenchrus and Vaccinium congeners, but failed to distinguish Solanum congeners or representatives of the Phaseolus vulgaris major gene pools. The ability of primer sets to amplify a wider range of plant species' introns and the presence of intron polymorphisms that distinguish congeners was confirmed by in silico analysis. While mitochondrial intron variation is limited in comparison to nuclear introns, these exon-based primer sets provide robust tools for the amplification of mitochondrial introns across a wide range of plant species wherein useful polymorphisms can be identified.
ABSTRACT
Viroids are RNA-based infectious agents that are single-stranded, covalently closed circular, non-coding, and naked. Unlike RNA viruses, which at least encode proteins for replication, encapsidation, and movement, lack of protein-coding capacity of viroids makes them completely reliant on host for replication and movement. The high genetic diversity in viroids is believed to be due to the absence of proof-reading activity of the host RNA polymerases, the large population size, and the rapid rate of replication. Protoplasts are viable plant cells that are prepared by enzymatic removal of cell walls. Plant protoplasts provide a synchronous single-cell system for studying early events of viroid infection such as replication and genetic diversity at the cellular level. A simple and efficient method to isolate and transfect citrus protoplasts with transcript RNA of citrus exocortis viroid is described in this chapter.
Subject(s)
Citrus , Viroids , Citrus/genetics , Plant Diseases , Plants , Protoplasts , RNA , RNA, Viral/genetics , Transfection , Viroids/geneticsABSTRACT
With the high demand for a healthy diet, it is necessary and important to explore natural sweeteners used in food that enhance palatability but minimize calories. Citrus is considered a good potential source of noncaloric sweeteners, but to date, only one sweetness modulator has been found in this most common fruit crop. Herein, an efficient strategy based on an in-house database and the untargeted and targeted metabolomics analyses was proposed to screen sweeteners or sweet-enhancing compounds from citrus. Eight sweeteners or sweetness-enhancing compounds were screened out, seven of which were newly identified from the genus Citrus. Surprisingly, we identified naturally occurring oxime V, which previously was only known as a synthetic compound. The contents of five compounds, in 11 citrus cultivars or unreleased selections across two production years, were compared. Successful identification of these natural sweeteners and sweetness-enhancing compounds in citrus fruit indicated the potential to identify the relevant biosynthetic pathways and to breed new citrus cultivars containing these compounds that provided both palatability and lower sugar consumption. This study also demonstrated that the proposed metabolomics-based screening strategy could greatly boost the identification of taste modulators with low contents in natural resources.
Subject(s)
Citrus , Sweetening Agents , Metabolomics , Plant Breeding , TasteABSTRACT
BACKGROUND: The genetic engineering of crops has enhanced productivity in the face of climate change and a growing global population by conferring desirable genetic traits, including the enhancement of biotic and abiotic stress tolerance, to improve agriculture. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system has been found to be a promising technology for genomic editing. Protoplasts are often utilized for the development of genetically modified plants through in vitro integration of a recombinant DNA fragment into the plant genome. We targeted the citrus Nonexpressor of Pathogenesis-Related 3 (CsNPR3) gene, a negative regulator of systemic acquired resistance (SAR) that governs the proteasome-mediated degradation of NPR1 and developed a genome editing technique targeting citrus protoplast DNA to produce stable genome-edited citrus plants. RESULTS: Here, we determined the best cationic lipid nanoparticles to deliver donor DNA and described a protocol using Lipofectamine™ LTX Reagent with PLUS Reagent to mediate DNA delivery into citrus protoplasts. A Cas9 construct containing a gRNA targeting the CsNPR3 gene was transfected into citrus protoplasts using the cationic lipid transfection agent Lipofectamine with or without polyethylene glycol (PEG, MW 6000). The optimal transfection efficiency for the encapsulation was 30% in Lipofectamine, 51% in Lipofectamine with PEG, and 2% with PEG only. Additionally, plasmid encapsulation in Lipofectamine resulted in the highest cell viability percentage (45%) compared with PEG. Nine edited plants were obtained and identified based on the T7EI assay and Sanger sequencing. The developed edited lines exhibited downregulation of CsNPR3 expression and upregulation of CsPR1. CONCLUSIONS: Our results demonstrate that utilization of the cationic lipid-based transfection agent Lipofectamine is a viable option for the successful delivery of donor DNA and subsequent successful genome editing in citrus.
ABSTRACT
Citrus is a major fruit crop cultivated on a global scale. Citrus trees are long lived perennials with a large canopy. Understanding the genetic control of tree architecture could provide tools for breeding and selection of citrus cultivars suitable for high density planting with improved light exposure. Tree architecture is modulated by the TILLER ANGLE CONTROL 1 (TAC1) gene which plays an important role in the regulation of the shoot angle. Herein, we used CRISPR/Cas9 technology to knockout the CsTAC1 gene for the biochemical and molecular analysis of its function. Nine transgenic lines were obtained, and five edited plants were confirmed based on T7EI mismatch detection assay and Sanger sequencing. The transgenic citrus lines exhibited pleiotropic phenotypes, including differences in branch angle and stem growth. Additionally, silencing CsTAC1 led to enhanced CsLAZY1 transcript levels in the tested lines. Analysis of the phytohormonal profile revealed that TAC1-edited plants exhibited lower auxin contents and increased cytokinin levels in the leaves compared to the wild-type plants. The GA7 gibberellin level was enhanced in most of the edited lines. Collectively, TAC1 affects branch angle in association with hormone signals in citrus.
Subject(s)
Citrus sinensis , Citrus , Citrus/metabolism , Citrus sinensis/genetics , Citrus sinensis/metabolism , Gene Expression Regulation, Plant , Hormones , Plant Breeding , Plant Proteins/genetics , Plant Proteins/metabolism , Trees/metabolismABSTRACT
The Australian finger lime (Citrus australasica) is tolerant to Huanglongbing (HLB; Citrus greening). This species can be utilized to develop HLB tolerant citrus cultivars through conventional breeding and biotechnological approaches. In this report, we conducted a comprehensive analysis of transcriptomic data following a non-choice infection assay to understand the CaLas tolerance mechanisms in the finger lime. After filtering 3,768 differentially expressed genes (DEGs), 2,396 were downregulated and 1,372 were upregulated in CaLas-infected finger lime compared to CaLas-infected HLB-susceptible 'Valencia' sweet orange. Comparative analyses revealed several DEGs belonging to cell wall, ß-glucanase, proteolysis, R genes, signaling, redox state, peroxidases, glutathione-S-transferase, secondary metabolites, and pathogenesis-related (PR) proteins categories. Our results indicate that the finger lime has evolved specific redox control systems to mitigate the reactive oxygen species and modulate the plant defense response. We also identified candidate genes responsible for the production of Cys-rich secretory proteins and Pathogenesis-related 1 (PR1-like) proteins that are highly upregulated in infected finger lime relative to noninfected and infected 'Valencia' sweet orange. Additionally, the anatomical analysis of phloem and stem tissues in finger lime and 'Valencia' suggested better regeneration of phloem tissues in finger lime in response to HLB infection. Analysis of callose formation following infection revealed a significant difference in the production of callose plugs between the stem phloem of CaLas+ 'Valencia' sweet orange and finger lime. Understanding the mechanism of resistance will help the scientific community design strategies to protect trees from CaLas infection and assist citrus breeders in developing durable HLB tolerant citrus varieties.