Generation of transgene-free canker-resistant Citrus sinensis cv. Hamlin in the T0 generation through Cas12a/CBE co-editing.

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.

Generation of the transgene-free canker-resistant Citrus sinensis using Cas12a/crRNA ribonucleoprotein in the T0 generation.

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.

Plant mitochondrial introns as genetic markers - conservation and variation.

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.

Effects of rootstocks on the flavor quality of huanglongbing-affected sweet orange juices using targeted flavoromics strategy.

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.

Effects of Different Rootstocks on the Metabolites of Huanglongbing-Affected Sweet Orange Juices Using a Novel Combined Strategy of Untargeted Metabolomics and Machine Learning.

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.

Susceptibility of Novel Promising Citrus Rootstocks to White Root Rot.

Citrus is one of the most important fruit crops in Mediterranean countries such as Spain, which is one of the main citrus-producing countries worldwide. Soil-borne pathogens, such as Rosellinia necatrix, are relevant limiting biotic factors in fruit trees, due to their tricky management. This fungus is a polyphagous plant pathogen with worldwide distribution, causing white root rot in woody crops, including citrus trees in Spain. The objective of this study was to evaluate the tolerance of new plant material against R. necatrix infection. Therefore, plants of 12 different citrus rootstocks were inoculated with one R. necatrix isolate. During the assay, and periodically, above-ground symptoms and chlorophyll content were evaluated. At the end of the experiment, leaf area and plant biomass measures were obtained. Rootstocks B11R5T64 and B11R5T60 achieved the lowest disease incidence of symptoms and reduction of biomass, and were similar to their respective controls in chlorophyll content and leaf area. Carrizo citrange, CL-5146 and UFR-5 were the most affected rootstocks in symptoms and biomass reduction. This work provides information about R. necatrix-tolerant citrus rootstocks, which can constitute a new integrated, sustainable and effective long-term strategy to avoid white root rot.

Insights into the mechanism of Huanglongbing tolerance in the Australian finger lime (Citrus australasica).

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.

Overexpression of the salicylic acid binding protein 2 (SABP2) from tobacco enhances tolerance against Huanglongbing in transgenic citrus.

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.

Acetylome reprograming participates in the establishment of fruit metabolism during polyploidization in citrus.

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.

Natural Sweeteners and Sweetness-Enhancing Compounds Identified in Citrus Using an Efficient Metabolomics-Based Screening Strategy.

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.

The Citrus sinensis TILLER ANGLE CONTROL 1 (CsTAC1) gene regulates tree architecture in sweet oranges by modulating the endogenous hormone content.

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.

A cationic lipid mediated CRISPR/Cas9 technique for the production of stable genome edited citrus plants.

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.

Utilization of somatic fusion techniques for the development of HLB tolerant breeding resources employing the Australian finger lime (Citrus australasica).

The Australian finger lime is a unique citrus species that has gained importance due to its unique fruit characteristics and perceived tolerance to Huanglongbing (HLB), an often-fatal disease of citrus trees. In this study, we developed allotetraploid finger lime hybrids and cybrids by utilizing somatic cell fusion techniques to fuse diploid 'OLL8' sweet orange or 'Page' tangelo callus-derived protoplasts with finger lime (FL) mesophyll-derived protoplasts. Six somatic fusions were regenerated from the 'OLL8' + FL fusion, while three putative cybrids were regenerated from the 'Page' + FL fusion. Ploidy levels and nuclear-expressed sequence tag derived simple sequence repeat (EST-SSR) markers confirmed the somatic hybrid production, and mitochondrial DNA primer sets confirmed the cybrid nature. Several trees produced by the somatic fusion remained HLB negative even after 6 years of growth in an HLB-endemic environment. Pathogenesis related (PR) and other genes that are often upregulated in HLB-tolerant trees were also upregulated in our somatic fusions. These newly developed somatic fusions and cybrids could potentially be used as breeding parents to develop the next generation of improved HLB-tolerant rootstocks and scions.

Physiological Responses and Gene Expression Patterns in Open-Pollinated Seedlings of a Pummelo-Mandarin Hybrid Rootstock Exposed to Salt Stress and Huanglongbing.

Huanglongbing (HLB), caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus (CaLas), is the primary biotic stress causing significant economic damage to the global citrus industry. Among the abiotic stresses, salinity affects citrus production worldwide, especially in arid and coastal regions. In this study, we evaluated open-pollinated seedlings of the S10 (a diploid rootstock produced from a cross between two siblings of the Hirado Buntan Pink pummelo (Citrus maxima (Burm.) Merr.) with the Shekwasha mandarin (Citrus reticulata Blanco)) for their ability to tolerate HLB and salinity stresses. In a greenhouse study, 'Valencia' sweet orange (either HLB-positive or negative) was grafted onto six clonally propagated lines generated from the screened seedlings in the greenhouse and the trees were irrigated with 150 mM NaCl after eight months of successful grafting and detection of CaLas in the leaf petioles. Cleopatra mandarin was used as a salt-tolerant and HLB-sensitive rootstock control. CaLas infection was monitored using a quantitative polymerase chain reaction before and after NaCl treatments. Following three months of NaCl treatment, 'Valencia' leaves on the S10 rootstock seedlings recorded lower levels of chlorophyll content compared to Cleopatra under similar conditions. Malondialdehyde content was higher in HLB-infected 'Valencia' grafted onto Cleopatra than in the S10 lines. Several plant defense-related genes were significantly upregulated in the S10 lines. Antioxidant and Na+ co-transporter genes were differentially regulated in these lines. Based on our results, selected S10 lines have potential as salt-tolerant rootstocks of 'Valencia' sweet orange under endemic HLB conditions. However, it is necessary to propagate selected lines through tissue culture or cuttings because of the high percentage of zygotic seedlings derived from S10.

Metabolic Profiling of Hybrids Generated from Pummelo and Citrus latipes in Relation to Their Attraction to Diaphorina citri, the Vector of Huanglongbing.

The citrus industry at present is severely affected by huanglongbing disease (HLB). HLB is caused by the supposed bacterial pathogen "Candidatus Liberibacter asiaticus" and is transmitted by the insect vector, the Asian citrus psyllid, Diaphorina citri Kuwayama. Developing new citrus hybrids to improve HLB management is much needed. In this study, we investigated the metabolomic profiles of three new hybrids produced from the cross of C2-5-12 Pummelo (Citrus maxima (L.) Osbeck) × pollen from Citrus latipes. The hybrids were selected based on leaf morphology and seedling vigor. The selected hybrids exhibited compact and upright tree architecture as seen in C. latipes. Hybrids were verified by simple sequence repeat markers, and were subjected to metabolomic analysis using gas chromatography-mass spectrometry. The volatile organic compounds (VOCs) and polar metabolites profiling also showed that the new hybrids were different from their parents. Interestingly, the levels of stored VOCs in hybrid II were higher than those observed in its parents and other hybrids. The level of most VOCs released by hybrid II was also higher than that released from its parents. Additionally, the preference assay showed that hybrid II was more attractive to D. citri than its parents and other hybrids. The leaf morphology, compact and upright architecture of hybrid II, and its attraction to D. citri suggest that it could be used as a windbreak and trap tree for D. citri (double duty), once its tolerance to HLB disease is confirmed. Our results showed that metabolomic analysis could be successfully used to understand the biochemical mechanisms controlling the interaction of D. citri with its host plants.

The vascular targeted citrus FLOWERING LOCUS T3 gene promotes non-inductive early flowering in transgenic Carrizo rootstocks and grafted juvenile scions.

Shortening the juvenile stage in citrus and inducing early flowering has been the focus of several citrus genetic improvement programs. FLOWERING LOCUS T (FT) is a small phloem-translocated protein that regulates precocious flowering. In this study, two populations of transgenic Carrizo citrange rootstocks expressing either Citrus clementina FT1 or FT3 genes under the control of the Arabidopsis thaliana phloem specific SUCROSE SYNTHASE 2 (AtSUC2) promoter were developed. The transgenic plants were morphologically similar to the non-transgenic controls (non-transgenic Carrizo citrange), however, only AtSUC2-CcFT3 was capable of inducing precocious flowers. The transgenic lines produced flowers 16 months after transformation and flower buds appeared 30-40 days on juvenile immature scions grafted onto transgenic rootstock. Gene expression analysis revealed that the expression of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1) and APETALA1 (AP1) were enhanced in the transgenics. Transcriptome profiling of a selected transgenic line showed the induction of genes in different groups including: genes from the flowering induction pathway, APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) family genes, and jasmonic acid (JA) pathway genes. Altogether, our results suggested that ectopic expression of CcFT3 in phloem tissues of Carrizo citrange triggered the expression of several genes to mediate early flowering.

Efficient CRISPR/Cas9 genome editing with Citrus embryogenic cell cultures.

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.

Localization and characterization of Citrus centromeres by combining half-tetrad analysis and CenH3-associated sequence profiling.

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.

All roads lead to Rome: Towards understanding different avenues of tolerance to huanglongbing in citrus cultivars.

Citrus tolerance to huanglongbing could result from tolerance to the pathogen Candidatus Liberibacter asiaticus (CLas) and/or to its vector Diaphorina citri. Field observations and greenhouse-controlled studies showed that some citrus cultivars were more tolerant than others. However, the mechanism(s) behind the tolerance has not been determined yet. Using GC-MS, we investigated the volatile organic compounds (VOCs) and the non-volatile metabolite profiles of two tolerant citrus cultivars- Australian finger lime, 'LB8-9' Sugar Belle® mandarin hybrid, and a recently released mandarin hybrid 'Bingo'. The three were grafted onto the rootstock, Carrizo citrange. Our findings showed that the metabolomic profiles of Australian finger lime were different from that of 'LB8-9'. Finger lime was high in many amino acids and tricarboxylic acid intermediates, whereas 'LB8-9' was high in several amino acids, sugars, and sugar alcohols. 'LB8-9' was high in thymol, which is known for its strong antimicrobial activity against a panel of pathogenic bacteria. The metabolomic profiles of 'Bingo' were intensely different from the other mandarin hybrid, 'LB8-9', including a reduced thymol biosynthetic pathway and low amounts of most of the amino acids and sugar alcohols. Remarkably, 1,8-cineole (eucalyptol) was only detected in 'Bingo', indicating that eucalyptol could have feeding and ovipositional repellency against D. citri. The metabolite profiles generated for HLB-tolerant citrus species will improve the ability of citrus breeders and will allow them to take more informed decisions. Metabolomic profiling of HLB-tolerant citrus species could identify tolerance specific markers that can be introduced to other commercial citrus cultivars to improve their tolerance to HLB disease.

Enhanced resistance to citrus canker in transgenic mandarin expressing Xa21 from rice.

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.