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Citrus bacterial canker (CBC) is resulted from Xanthomonas citri subsp. citri (Xcc) infection and poses a significant threat to citrus production. Glutathione S-transferases (GSTs) are critical in maintaining redox homeostasis in plants, especially in relation to abiotic and biotic stress responses. However, the function of GSTs in resisting CBC remains unclear. Here, citrus glutathione S-transferases were investigated applying a genome-wide approach. In total, 69 CsGSTs belonging to seven classes were identified, and the phylogeny, chromosomal distribution, gene structures and conserved motifs were analyzed. Several CsGSTs responded to Xcc infection, as observed in the upregulation of CsGSTF1 and CsGSTU18 in the CBC-sensitive ‘Wanjincheng’ variety but not in the resistant ‘Kumquat’ variety. CsGSTF1 and CsGSTU18 were localized at the cytoplasm. Transient overexpression of CsGSTF1 and CsGSTU18 mediated reactive oxygen species (ROS) scavenging, whereas the virus-induced gene silencing (VIGS) of CsGSTF1 and CsGSTU18 caused strong CBC resistance and ROS burst. The present study investigated the characterization of citrus GST gene family, and discovered that CsGSTF1 and CsGSTU18 negatively contributed to CBC through modulating ROS homeostasis. These findings emphasize the significance of GSTs in infection resistance in plants.

Huanglongbing (HLB) and citrus canker, arising from Candidatus Liberibacter asiaticus (CaLas) and Xanthomonas citri pv. Citri (Xcc), respectively, have been imposing tremendous losses to the global citrus industry. Systemic acquired resistance (SAR) has been shown to be crucial for priming defense against pathogen in citrus. Salicylic acid (SA) binding protein 2 (SABP2), which is responsible for converting methyl salicylate (MeSA) to SA, is essential for full SAR establishment. Here, we characterized the functions of four citrus SABP2 genes (CsSABP2-1, CsSABP2-1V18A, CsSABP2-2 and CsSABP2-3) against HLB and citrus canker. In vitro enzymatic assay revealed that all four proteins had MeSA esterase activities, and CsSABP2-1 and CsSABP2-1V18A has the strongest activity. Their activities were inhibited by SA except for CsSABP2-1V18A. Four genes controlled by a strong promoter 35S were induced into Wanjincheng orange (Citrus sinensis Osbeck) to generate transgenic plants overexpressing CsSABP2. Overexpressing CsSABP2 increased SA and MeSA content and CsSABP2-1V18A had the strongest action on SA. Resistance evaluation demonstrated that only CsSABP2-1V18A had significantly enhanced tolerance to HLB, although all four CsSABP2s had increased tolerance to citrus canker. The data suggested the amino acid Val-18 in the active site of CsSABP2 plays a key role in protein function. Our study emphasized that balancing the levels of SA and MeSA is crucial for regulating SAR and conferring broad-spectrum resistance to HLB and citrus canker. This finding offers valuable insights for enhancing resistance through SAR engineering.

One of the main diseases that adversely impacts the global citrus industry is citrus bacterial canker (CBC), caused by the bacteria Xanthomonas citri subsp. citri (Xcc). Response to CBC is a complex process, with both protein-DNA as well as protein–protein interactions for the regulatory network. To detect such interactions in CBC resistant regulation, a citrus high-throughput screening system with 203 CBC-inducible transcription factors (TFs), were developed. Screening the upstream regulators of target by yeast-one hybrid (Y1H) methods was also performed. A regulatory module of CBC resistance was identified based on this system. One TF (CsDOF5.8) was explored due to its interactions with the 1-kb promoter fragment of CsPrx25, a resistant gene of CBC involved in reactive oxygen species (ROS) homeostasis regulation. Electrophoretic mobility shift assay (EMSA), dual-LUC assays, as well as transient overexpression of CsDOF5.8, further validated the interactions and transcriptional regulation. The CsDOF5.8–CsPrx25 promoter interaction revealed a complex pathway that governs the regulation of CBC resistance via H2O2 homeostasis. The high-throughput Y1H/Y2H screening system could be an efficient tool for studying regulatory pathways or network of CBC resistance regulation. In addition, it could highlight the potential of these candidate genes as targets for efforts to breed CBC-resistant citrus varieties.

Abstract Citrus Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CaLas), is the most serious disease worldwide. CaLasSDE460 was previously characterized as a potential virulence factor of CaLas. However, the function and mechanism of CaLasSDE460 involved in CaLas against citrus is still elusive. Here, we showed that transgenic expression of CaLasSDE460 in Wanjincheng oranges (C. sinensis Osbeck) contributed to the early growth of CaLas and the development of symptoms. When the temperature increased from 25 °C to 32 °C, CaLas growth and symptom development in transgenic plants were slower than those in WT controls. RNA-seq analysis of transgenic plants showed that CaLasSDE460 affected multiple biological processes. At 25 °C, transcription activities of the “Protein processing in endoplasmic reticulum” and “Cyanoamino acid metabolism” pathways increased while transcription activities of many pathways decreased at 32 °C. 124 and 53 genes, separately annotated to plant-pathogen interaction and MAPK signaling pathways, showed decreased expression at 32 °C, compared with these (38 for plant-pathogen interaction and 17 for MAPK signaling) at 25 °C. Several important genes (MAPKKK14, HSP70b, NCED3 and WRKY33), remarkably affected by CaLasSDE460, were identified. Totally, our data suggested that CaLasSDE460 participated in the pathogenesis of CaLas through interfering transcription activities of citrus defense response and this interfering was temperature-dependent. Graphical Abstract

Genetic transformation with mature material as the explants could shorten the transgenic period and avoid seed dependence compared with genetic transformation using the epicotyl seedling stem segments as the receptor. Here, we constructed an Agrobacterium tumefaciens-mediated transformation for generation of marker-free transgenic plants from navel orange (Citrus sinensis Osbeck) mature stems using a Cre-loxP recombination system. To efficiently recover the regenerated buds from mature tissues, five recovery methods were compared: in vitro micrografting of 0.1–0.5 (1–2 weeks), > 0.5 cm (3–4 weeks) and > 1 cm long lignified bud and in vitro micrografting of explants with a bud and rooting regenerated bud. The data showed that in vitro micrografting of > 1 cm long regenerated bud with expanded leaves after one month of continuous culture for lignification was the optimal solution for plant recovery from mature tissues. Transgenic plants without selectable marker genes were created from navel orange (Citrus sinensis Osbeck) tissue using a transformation vector PLI-35SPR1aCB containing a Cre/loxP system recombination together with genes encoding the selectable marker isopentenyl transferase (IPT) and an anti-bacterial peptide (PR1aCB). Using IPT positive selection, the transformation efficiency determined by PCR was 0.9%, and in total, 20 transgenic plants were obtained. Southern blotting confirmed further their transgenicity. PCR and sequencing analysis demonstrated that both the Cre and IPT genes had been successfully removed from the transgenic plants (deletion efficiency 100%). Over all, using Cre/loxP system recombination together with the IPT positive selection, marker-free transgenic plants can be recovered efficiently from mature tissues of navel orange (Citrus sinensis Osbeck), which provides a potential method for production of transgenic plants from citrus mature tissue.

BackgroundCopy number variations (CNVs) have been shown to be overrepresented in children with congenital heart disease (CHD). Genetic evaluation of CHD is currently underperformed in China. We sought to determine the occurrence of CNVs in CNV regions with disease-causing potential among a large cohort of Chinese pediatric CHD patients and investigate whether these CNVs could be the important critical modifiers of surgical intervention.MethodsCNVs screenings were performed in 1,762 Chinese children who underwent at least one cardiac surgery. CNV status at over 200 CNV locus with disease-causing potential was analyzed with a high-throughput ligation-dependent probe amplification (HLPA) assay.ResultsWe found 378 out of 1,762 samples (21.45%) to have at least one CNV and 2.38% of them were carrying multiple CNVs. The detection rates of ppCNVs (pathogenic and likely pathogenic CNVs) were 9.19% (162/1,762), significantly higher than that of the healthy Han Chinese individuals from The Database of Genomic Variants archive (9.19% vs. 3.63%; P = 0.0012). CHD cases with ppCNVs had a significantly higher proportion of complex surgeries compared to CHD patients with no ppCNVs (62.35% vs. 37.63%, P

Huanglongbing (HLB), caused by “Candidatus liberibacter asiaticus” (CaLas), is one of the most devastating diseases in citrus but its pathogenesis remains poorly understood. Here, we reported the role of the CaLasSDE115 (CLIBASIA_05115) effector, encoded by CaLas, during pathogen-host interactions. Bioinformatics analyses showed that CaLasSDE115 was 100% conserved in all reported CaLas strains but had sequence differences compared with orthologs from other “Candidatus liberibacter.” Prediction of protein structures suggested that the crystal structure of CaLasSDE115 was very close to that of the invasion-related protein B (IalB), a virulence factor from Bartonella henselae. Alkaline phosphatase (PhoA) assay in E. coli further confirmed that CaLasSDE115 was a Sec-dependent secretory protein while subcellular localization analyses in tobacco showed that the mature protein of SDE115 (mSDE115), without its putative Sec-dependent signal peptide, was distributed in the cytoplasm and the nucleus. Expression levels of CaLasSDE115 in CaLas-infected Asian citrus psyllid (ACP) were much higher (∼45-fold) than those in CaLas-infected Wanjincheng oranges, with the expression in symptomatic leaves being significantly higher than that in asymptomatic ones. Additionally, the overexpression of mSDE115 favored CaLas proliferation during the early stages (2 months) of infection while promoting the development of symptoms. Hormone content and gene expression analysis of transgenic plants also suggested that overexpressing mSDE115 modulated the transcriptional regulation of genes involved in systemic acquired resistance (SAR) response. Overall, our data indicated that CaLasSDE115 effector contributed to the early colonization of citrus by the pathogen and worsened the occurrence of Huanglongbing symptoms, thereby providing a theoretical basis for further exploring the pathogenic mechanisms of Huanglongbing disease in citrus.

Superoxide dismutases (SODs) play critical roles in plants, especially in the maintenance of redox homeostasis. The response of SODs in Citrus (Citrus sinensis (L.) Osbeck) to citrus bacterial canker (CBC) infection were investigated. The CsSODs were identified, and their gene structures, phylogeny, conserved domains and motifs, predicted interactions, and chromosomal distribution were analyzed. CsSOD expression in response to stress-related plant hormones (salicylic acid, SA; methyl jasmonate, MeJA; and abscisic acid, ABA) and Xanthomonas citri subsp. citri (Xcc) infection were also investigated. Thirteen CsSODs were identified in C. sinensis, including four Fe/MnSODs and nine Cu/ZnSODs with typical functional domains. The CsSODs were distributed on chromosomes 3, 5, 7, and 8. Specific hormone-response motifs were identified in the gene promoter regions. Ten genes were induced by MeJA treatment, as shown by qRT-PCR, and were upregulated in the CBC-susceptible Wanjincheng citrus variety, while CsSOD06 and CsSOD08 were upregulated by ABA in both the Wanjincheng and the CBC-resistant Kumquat varieties. Xcc infection significantly altered the levels of most CsSODs. The overexpression of CsSOD06 and CsSOD08 resulted in increased hydrogen peroxide levels and SOD activity. Our findings highlight the significance of SOD enzymes in the plant response to pathogen infection and have a potential application for breeding CBC-tolerant citrus varieties.

Antagonism between jasmonic acid (JA) and salicylic acid (SA) plays pivotal roles in the fine-tuning of plant immunity against pathogen infection. In this study, we compared the phytohormonal responses to Xanthomonas citri subsp. citri (Xcc) between the citrus canker-susceptible (S) cultivar Wanjincheng orange (Citrus sinensis Osbeck) and -resistant (R) cultivar Jindan (Fortunella crassifolia Swingle). Upon Xcc infection, SA and JA were strongly induced in Jindan (R) and Wanjincheng orange (S), respectively, and JA appeared to contribute to citrus disease susceptibility by antagonizing SA-mediated effective defenses. A homologous gene encoding the allene oxide synthase (AOS) 1-2 enzyme, which catalyzes the first committed step in JA biosynthesis, was specifically upregulated in Wanjincheng orange (S) but not in Jindan (R). A promoter sequence analysis showed that abscisic acid (ABA)-responsive elements are enriched in the AOS1-2 of Wanjincheng orange (S) but not in Jindan (R). Accordingly, ABA treatments could induce AOS1-2 expression and JA accumulation, leading to enhanced citrus disease susceptibility in Wanjincheng orange (S), while the synthesis inhibitor sodium tungstate had the opposite effects. Moreover, ABA was specifically induced by Xcc infection in Wanjincheng orange (S) but not in Jindan (R). Thus, Xcc appeared to hijack host ABA biosynthesis to promote JA accumulation, which in turn suppressed effectual SA-mediated defenses to favor disease development in citrus. Our findings provide new insights into the molecular mechanisms underlying the differential citrus-canker resistance in citrus cultivars, and a new strategy for the biotechnological improvement of citrus canker resistance was discussed.

In this study, we performed a comprehensive survey of xyloglucan endotransglucosylase/hydrolase (XTH) and a functional validation of Citrus sinensis (Cs) XTH genes to provide new insights into the involvement of XTHs in Xanthomonas citri subsp. citri (Xcc) infection. From the genome of sweet orange, 34 CsXTH genes with XTH characteristic domains were identified and clustered into groups I/II, IIIA, and IIIB. Except for chromosome 9, the CsXTH genes were unevenly distributed and duplicated among all chromosomes, identifying a CsXTH duplication hot spot on chromosome 4. With Xcc induction, a group of citrus canker-related CsXTHs were detected. CsXTH04 was identified as a putative candidate gene, which is up-regulated in citrus bacterial canker (CBC)-resistant varieties and induced by exogenous treatment with salicylic acid (SA) and methyl jasmonate (MeJA). CsXTH04 overexpression conferred CBC susceptibility to transgenic citrus, while CsXTH04 silencing conferred CBC resistance. Taken together, the annotation of the CsXTH family provides an initial basis for the functional and evolutionary study of this family as potential CBC-susceptible genes. CsXTH04, validated in this study, can be used in citrus breeding to improve CBC resistance.

Citrus bacterial canker (CBC) caused by Xanthomonas citri subsp. citri (Xcc) is a systemic bacterial disease that affects citrus plantations globally. Biotic stress in plants has been linked to a group of important transcription factors known as Basic Leucine Zippers (BZIPs). In this study, CsBZIP40 was functionally characterized by expression analysis, including induction by Xcc and hormones, subcellular localization, over-expression and RNAi silencing. CsBZIP40 belongs to group D of the CsBZIP family of transcription factors and localizes in the nucleus, potentially serving as a transcriptional regulator. In wild type (WT) plants CsBZIP40 can be induced by plant hormones in addition to infection by Xcc which has given insight into its involvement in CBC. In the present study, over-expression of CsBZIP40 conferred resistance to Xcc while its silencing led to Xcc susceptibility. Both over-expression and RNAi affected salicylic acid (SA) production and expression of the genes involved in the SA synthesis and signaling pathway, in addition to interaction of CsBZIP40 with CsNPR1, as detected by a GST pull-down assay. Taken together, the results of this study confirmed the important role of CsBZIP40 in improving resistance to citrus canker through the SA signaling pathway by the presence of NPR1 to activate PR genes. Our findings are of potential value in the breeding of tolerance to CBC in citrus fruits.

The auxin early response gene Gretchen Hagen3 (GH3) plays dual roles in plant development and responses to biotic or abiotic stress. It functions in regulating hormone homeostasis through the conjugation of free auxin to amino acids. In citrus, GH3.1 and GH3.1L play important roles in responding to Xanthomonas citri subsp. citri (Xcc). Here, in Wanjingcheng orange (Citrus sinensis Osbeck), the overexpression of CsGH3.1 and CsGH3.1L caused increased branching and drooping dwarfism, as well as smaller, thinner and upward curling leaves compared with wild-type. Hormone determinations showed that overexpressing CsGH3.1 and CsGH3.1L decreased the free auxin contents and accelerated the Xcc-induced decline of free auxin levels in transgenic plants. A resistance analysis showed that transgenic plants had reduced susceptibility to citrus canker, and a transcriptomic analysis revealed that hormone signal transduction-related pathways were significantly affected by the overexpression of CsGH3.1 and CsGH3.1L. A MapMan analysis further showed that overexpressing either of these two genes significantly downregulated the expression levels of the annotated auxin/indole-3-acetic acid family genes and significantly upregulated biotic stress-related functions and pathways. Salicylic acid, jasmonic acid, abscisic acid, ethylene and zeatin levels in transgenic plants displayed obvious changes compared with wild-type. In particular, the salicylic acid and ethylene levels involved in plant resistance responses markedly increased in transgenic plants. Thus, the overexpression of CsGH3.1 and CsGH3.1L reduces plant susceptibility to citrus canker by repressing auxin signaling and enhancing defense responses. Our study demonstrates auxin homeostasis' potential in engineering disease resistance in citrus.

To study the functions of the early auxin-responsive genes CsGH3.1 and CsGH3.6 in citrus resistance against canker disease, we cloned CsGH3.1 and CsGH3.6 in ‘Newhall’ Navel Orange (Citrus sinensis Osbeck). They are 1 797 bp and 1 887 bp and encode 598 and 629 amino acids, respectively. In vitro mature leaves from susceptible ‘Newhall’ and resistant Calamondin (C. madurensis) were inoculated by a Xanthomonas axonopodis pv. citri (Xac) bacterial suspension, and expression of CsGH3.1 and CsGH3.6 in the two varieties were analyzed using quantitative real-time PCR (qRT-PCR). ‘Newhall’ leaves were treated with different hormones for 3 days, inoculated by Xac bacterial suspension, and then the symptoms in these leaves were investigated. We used qRT-PCR to analyze the effect of different hormones on CsGH3.1 and CsGH3.6 expression in ‘Newhall’ leaves. The expression levels of both CsGH3.1 and CsGH3.6 were significantly induced by Xac in ‘Newhall’ leaves, compared with levels in Calamondin leaves. 1-naphthy acetic acid (NAA) increased the hypertrophy of infection sites in ‘Newhall’ leaves, while naphthyl-phthalamic acid (NPA) had no visible effect on lesion development. NAA hormone greatly improved expression of CsGH3.1 in ‘Newhall’, but not CsGH3.6. These results indicate that the auxin primary-response gene CsGH3.1 plays an important role in citrus susceptibility to Xac.

Eucalyptus is a worldwide hard-wood species which increasingly focused on. To adapt to various biotic and abiotic stresses, Eucalyptus have evolved complex mechanisms, increasing the cellular concentration of reactive oxygen species (ROS) by numerous ROS controlling enzymes. To better analyse the ROS gene network and discuss the differences between four Eucalyptus species, ROS gene network including 11 proteins families (1CysPrx, 2CysPrx, APx, APx-R, CIII Prx, Diox, GPx, Kat, PrxII, PrxQ and Rboh) were annotated and compared in an expert and exhaustive manner from the genomic data available from E. camaldulensis, E. globulus, E. grandis, and E. gunnii. In addition, a specific sequencing strategy was performed in order to determine if the missed sequences in at least one organism are the results of gain/loss events or only sequencing gaps. We observed that the automatic annotation applied to multigenic families is the source of miss-annotation. Base on the family size, the 11 families can be categorized into duplicated gene families (CIII Prx, Kat, 1CysPrx, and GPx), which contain a lot of gene duplication events and non-duplicated families (APx, APx-R, Rboh, DiOx, 2CysPrx, PrxII, and PrxQ). The gene family sizes are much larger in Eucalyptus than most of other angiosperms due to recent gene duplications, which could give higher adaptability to environmental changes and stresses. The cross-species comparative analysis shows gene gain and loss events during the evolutionary process. The 11 families possess different expression patterns, while in the Eucalyptus genus, the ROS families present similar expression patterns. Overall, the comparative analysis might be a good criterion to evaluate the adaptation of different species with different characters, but only if data mining is as exhaustive as possible. It is also a good indicator to explore the evolutionary process.