[Determination of multiple restricted and prohibited ingredients in hair dyes using a novel 3D stationary phase coupled with gas chromatography-mass spectrometry].

Hair dyes (HDs) are mainly composed of various benzene series, amines, and phenolic compounds. These ingredients are well known to have allergenic, teratogenic, and carcinogenic properties. As such, the presence of these ingredients in HDs has received increased attention in recent years. At present, the applications of traditional analytical and detection methods and commercial chromatographic columns are limited by problems such as poor qualitative analysis and inaccurate quantification. Thus, the development of new analytical and detection technologies and stationary phases is an urgent endeavor. Moreover, HDs contain complex compounds and exhibit significant matrix interference. Hence, appropriate sample pretreatment methods are necessary to analyze HDs. In this study, the 3D nonpolar rigid structure of triptycene (TP) was combined with the polar flexible chains of polyethylene glycol (PEG) to design and synthesize a TP derivative, TP-PEG, as a stationary phase for chromatographic columns. The stationary phase enabled the expansion of the selection range for polar and nonpolar analytes. Subsequently, gas chromatography-mass spectrometry (GC-MS) was used to quantitatively analyze 22 ingredients in HDs. The experimental results demonstrated that analytes with different polarities exhibited sharp and symmetrical peak shapes on the stationary phase, and all 22 analytes achieved baseline separation on the chromatographic column. The 22 ingredients in HDs showed good linear relationships within their respective ranges, with correlation coefficients greater than 0.9985. The average recovery rates at three spiked levels were in the range of 89.2%-103.2%, and RSDs were less than 5%. Compared with traditional methods, the proposed method has higher efficiency and better accuracy, thus verifying the excellent separation performance of the new stationary phase and the effectiveness of the established GC-MS detection method. The findings indicated the applicability of the developed method to the detection and analysis of various compounds in HDs.

Systematic analysis and functional verification of citrus ascorbate peroxidases reveal that CsAPX01 and CsAPX02 negatively regulate citrus bacterial canker through the hydrogen peroxide regulation.

Ascorbate peroxidases (APXs) are antioxidant enzymes that play vital roles in redox homeostasis in plants. Citrus is susceptible to infection by Xanthomonas citri subsp. citri (Xcc), resulting in citrus bacterial canker (CBC). The present study used bioinformatic and expression analyses to investigate the APX family in Citrus sinensis. Bioinformatic research revealed the chromosomal locations, phylogeny, gene structure, promoter elements, functional domains, conserved motifs, and most likely physicochemical properties of the sequences. Six APXs clustered in three groups were identified, with each protein containing a single peroxidase domain. The promoter regions contained a variety of transcription factor-binding and hormone-response components. Xcc infection induced different CsAPX01 and CsAPX02 expressions in the CBC-susceptible Wanjincheng and CBC-resistant Kumquat varieties. Subcellular localization and transient expression showed that CsAPX01 and CsAPX02 were expressed in the cytoplasm and nucleus and had hydrogen peroxide (H2O2)-scavenging activity. Virus-induced gene silencing (VIGS) of CsAPX01 and CsAPX02 resulted in strong resistance to CBC and H2O2 bursts without effects on the plant phenotype. The current study focused on investigating and characterizing the citrus APX family. It was found that CsAPX01 and CsAPX02 exacerbated CBC by altering the balance of H2O2. These findings emphasize the importance of APXs in enhancing plant resistance to pathogens.

Rational design of a NIR fluorescent probe and its application in food detection of viscosity and biosystem imaging.

Viscosity is one of the most important parameters of liquid foods and shows significant change during food spoilage. It is also an important component of the cell microenvironment and is closely associated with the development of liver injury. In this work, a viscosity-sensitive fluorescent probe named WZ-V based on the twisted intramolecular charge transfer (TICT) mechanism was successfully designed. WZ-V had a large Stokes shift, long wavelength emission, and the fluorescence intensity shows 290-fold enhancement in high viscosity. Probe WZ-V successfully detected viscosity changes caused by food thickeners, as well as in milk, orange juice, and lemonade spoilage processes. This provides a new tool for regulating the viscosity of liquid foods and monitoring viscosity changes during food spoilage. In addition, WZ-V has been successfully applied to image viscosity changes in liver injury, which provides an important reference for the study of liver diseases.

The wall-associated receptor-like kinase CsWAKL01, positively regulated by the transcription factor CsWRKY53, confers resistance to citrus bacterial canker via regulation of phytohormone signaling.

Citrus bacterial canker (CBC) is a disease that poses a major threat to global citrus production and is caused by infection with Xanthomonas citri subsp. citri (Xcc). Wall-associated receptor-like kinase (WAKL) proteins play an important role in shaping plant resistance to various bacterial and fungal pathogens. In a previous report, CsWAKL01 was identified as a candidate Xcc-inducible gene found to be up-regulated in CBC-resistant citrus plants. However, the functional role of CsWAKL01 and the mechanisms whereby it may influence resistance to CBC have yet to be clarified. Here, CsWAKL01 was found to localize to the plasma membrane, and the overexpression of the corresponding gene in transgenic sweet oranges resulted in pronounced enhancement of CBC resistance, whereas its knockdown had the opposite effect. Mechanistically, the effect of CsWAKL01 was linked to its ability to reprogram jasmonic acid, salicylic acid, and abscisic acid signaling activity. CsWRKY53 was further identified as a transcription factor capable of directly binding to the CsWAKL01 promoter and inducing its transcriptional up-regulation. CsWRKY53 silencing conferred greater CBC susceptibility to infected plants. Overall, these data support a model wherein CsWRKY53 functions as a positive regulator of CsWAKL01 to enhance resistance to CBC via the reprogramming of phytohormone signaling. Together these results offer new insights into the mechanisms whereby WAKLs shape phytopathogen resistance while underscoring the potential value of targeting the CsWRKY53-CsWAKL01 axis when seeking to breed CBC-resistant citrus plant varieties.

Genome-wide identification and characterization of the sweet orange (Citrus sinensis) basic helix-loop-helix (bHLH) family reveals a role for CsbHLH085 as a regulator of citrus bacterial canker resistance.

Citrus bacterial canker (CBC) is a harmful bacterial disease caused by Xanthomonas citri subsp. citri (Xcc), negatively impacting citrus production worldwide. The basic helix-loop-helix (bHLH) transcription factor family plays crucial roles in plant development and stress responses. This study aimed to identify and annotate bHLH proteins encoded in the Citrus sinensis genome and explore their involvement and functional importance in regulating CBC resistance. A total of 135 putative CsbHLHs TFs were identified and categorized into 16 subfamilies. Their chromosomal locations, collinearity, and phylogenetic relationships were comprehensively analyzed. Upon Xcc strain YN1 infection, certain CsbHLHs were differentially regulated in CBC-resistant and CBC-sensitive citrus varieties. Among these, CsbHLH085 was selected for further functional characterization. CsbHLH085 was upregulated in the CBC-resistant citrus variety, was localized in the nucleus, and had a transcriptional activation activity. CsbHLH085 overexpression in Citrus significantly enhanced CBC resistance, accompanied by increased levels of salicylic acid (SA), jasmonic acid (JA), reactive oxygen species (ROS), and decreased levels of abscisic acid (ABA) and antioxidant enzymes. Conversely, CsbHLH085 virus-induced gene silencing resulted in opposite phenotypic and biochemical responses. CsbHLH085 silencing also affected the expression of phytohormone biosynthesis and signaling genes involved in SA, JA, and ABA signaling. These findings highlight the crucial role of CsbHLH085 in regulating CBC resistance, suggesting its potential as a target for biotechnological-assisted breeding citrus varieties with improved resistance against phytopathogens.

Genome-Wide Identification and Characterization of the Sweet Orange (Citrus sinensis) GATA Family Reveals a Role for CsGATA12 as a Regulator of Citrus Bacterial Canker Resistance.

Citrus bacterial canker (CBC) is a severe bacterial infection caused by Xanthomonas citri subsp. citri (Xcc), which continues to adversely impact citrus production worldwide. Members of the GATA family are important regulators of plant development and regulate plant responses to particular stressors. This report aimed to systematically elucidate the Citrus sinensis genome to identify and annotate genes that encode GATAs and evaluate the functional importance of these CsGATAs as regulators of CBC resistance. In total, 24 CsGATAs were identified and classified into four subfamilies. Furthermore, the phylogenetic relationships, chromosomal locations, collinear relationships, gene structures, and conserved domains for each of these GATA family members were also evaluated. It was observed that Xcc infection induced some CsGATAs, among which CsGATA12 was chosen for further functional validation. CsGATA12 was found to be localized in the nucleus and was differentially upregulated in the CBC-resistant and CBC-sensitive Kumquat and Wanjincheng citrus varieties. When transiently overexpressed, CsGATA12 significantly reduced CBC resistance with a corresponding increase in abscisic acid, jasmonic acid, and antioxidant enzyme levels. These alterations were consistent with lower levels of salicylic acid, ethylene, and reactive oxygen species. Moreover, the bacteria-induced CsGATA12 gene silencing yielded the opposite phenotypic outcomes. This investigation highlights the important role of CsGATA12 in regulating CBC resistance, underscoring its potential utility as a target for breeding citrus varieties with superior phytopathogen resistance.

ABA-CsABI5-CsCalS11 module upregulates Callose deposition of citrus infected with Candidatus Liberibacter asiaticus.

Huanglongbing (HLB) primarily caused by Candidatus Liberibacter asiaticus (CLas) has been threatening citrus production globally. Under HLB conditions, an excessive accumulation of the polysaccharide callose in citrus phloem occurs, leading to phloem blockage and starch accumulation in leaves. The callose production is controlled by callose synthases (CalS), which have multiple members within plants. However, the knowledge of callose production in the citrus upon infection with CLas is limited. In this study, we firstly identified 11 CalSs in the Citrus sinensis genome through bioinformatics and found the expression pattern of CsCalS11 exhibited a positive correlation with callose deposition in CLas-infected leaves (correlation coefficient of 0.77, P ≤ 0.05). Knockdown of CsCalS11 resulted in a reduction of callose deposition and starch accumulation in CLas-infected citrus. Interestingly, we observed significantly higher concentrations of abscisic acid (ABA) in HLB-infected citrus leaves compared to uninfected ones. Furthermore, the expressions of CsABI5, CsPYR, and CsSnRK2 in the ABA pathway substantially increased in citrus leaves upon CLas infection. Additionally, the expression of CsCalS11 was significantly upregulated in citrus leaves following the application of exogenous ABA. We confirmed that CsABI5, a pivotal component of the ABA signaling pathway, regulates CsCalS11 expression by binding to its promoter using yeast one-hybrid assay, dual luciferase assay, and transient expression in citrus leaves. In conclusion, our findings strongly suggest that the CsABI5-CsCalS11 module plays a crucial role in regulating callose deposition through the ABA signaling pathway during CLas infection. The results also revealed new function of the ABA signaling pathway in plants under biotic stress.

[The role of jasmonic acid in stress resistance of plants: a review].

Jasmonic acid (JA), a plant endogenously synthesized lipid hormone, plays an important role in response to stress. This manuscript summarized the biosynthesis and metabolism of JA and its related regulatory mechanisms, as well as the signal transduction of JA. The mechanism and regulatory network of JA in plant response to biotic and abiotic stresses were systematically reviewed, with the latest advances highlighted. In addition, this review summarized the signal crosstalk between JA and other hormones in regulating plant resistance to various stresses. Finally, the problems to be solved in the study of plant stress resistance mediated by JA were discussed, and the application of new molecular biological technologies in regulating JA signaling to enhance crop resistance was prospected, with the aim to facilitate future research and application of plant stress resistance.

The CsAP2-09-CsWRKY25-CsRBOH2 cascade confers resistance against citrus bacterial canker by regulating ROS homeostasis.

Citrus bacterial canker (CBC) is a serious bacterial disease caused by Xanthomonas citri subsp. citri (Xcc) that adversely impacts the global citrus industry. In a previous study, we demonstrated that overexpression of an Xcc-inducible apetala 2/ethylene response factor encoded by Citrus sinensis, CsAP2-09, enhances CBC resistance. The mechanism responsible for this effect, however, is not known. In the present study, we showed that CsAP2-09 targeted the promoter of the Xcc-inducible WRKY transcription factor coding gene CsWRKY25 directly, activating its transcription. CsWRKY25 was found to localize to the nucleus and to activate transcriptional activity. Plants overexpressing CsWRKY25 were more resistant to CBC and showed higher expression of the respiratory burst oxidase homolog (RBOH) CsRBOH2, in addition to exhibiting increased RBOH activity. Transient overexpression assays in citrus confirmed that CsWRKY25 and CsRBOH2 participated in the generation of reactive oxygen species (ROS) bursts, which were able to restore the ROS degradation caused by CsAP2-09 knockdown. Moreover, CsWRKY25 was found to bind directly to W-box elements within the CsRBOH2 promoter. Notably, CsRBOH2 knockdown had been reported previously to reduce the CBC resistance, while demonstrated in this study, CsRBOH2 transient overexpression can enhance the CBC resistance. Overall, our results outline a pathway through which CsAP2-09-CsWRKY25 transcriptionally reprograms CsRBOH2-mediated ROS homeostasis in a manner conducive to CBC resistance. These data offer new insight into the mechanisms and regulatory pathways through which CsAP2-09 regulates CBC resistance, highlighting its potential utility as a target for the breeding of CBC-resistant citrus varieties.

A turn-on fluorescent probe for detecting and bioimaging of HOCl in inflammatory and liver disease models.

Hypochlorous acid (HOCl) is a common reactive oxygen species (ROS) associated with the development of liver, tumor, inflammatory, and other diseases. In this work, the turn-on fluorescent probe named (WZ-HOCl) with a naphthalimide structure was designed and synthesized to detect endogenous HOCl in disease models. WZ-HOCl can achieve a fast response to HOCl with good linearity in the range of 0-45 µM (LOD = 147 nM). The application of WZ-HOCl in bioimaging was investigated by constructing a series of cellular disease models, and the results showed that WZ-HOCl could sensitively detect endogenous HOCl in inflammatory and liver disease models. It can also be used to differentiate between hepatocytes and hepatoma cells. WZ-HOCl will provide new methods and ideas for fluorescent probes in detecting drug-induced liver injury, alcoholic and non-alcoholic steatohepatitis, and some inflammation-related diseases.

Function and molecular mechanism analysis of CaLasSDE460 effector involved in the pathogenesis of "Candidatus Liberibacter asiaticus" in citrus.

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.

CsBZIP40 confers resistance against citrus bacterial canker by repressing CsWRKY43-CsPrx53/CsSOD13 cascade mediated ROS scavenging.

As the bacterial etiologic agent causing citrus bacterial canker (CBC), Xanthomonas citri subsp. citri (Xcc) seriously impacts citrus plantation and fruit production globally. In an earlier study, we demonstrated that CsBZIP40 can positively impact CBC resistance in the sweet orange (Citrus sinensis). However, the mechanistic basis for the protective benefits conferred by CsBZIP40 is yet to be delineated. Here, we show that CsBZIP40 positively regulates CBC resistance and reactive oxygen species (ROS) homeostasis in transgenic sweet orange overexpressing CsBZIP40. CsBZIP40 directly binds to the TGA-box of the CsWRKY43 promoter to repress its transcriptional activity. CsWRKY43 overexpression induces CBC susceptibility in transgenic sweet oranges. In contrast, its inhibition produces strong resistance to CBC. CsWRKY43 directly binds to the W-boxes of the CsPrx53 and CsSOD13 promoters to positively regulate the activities of these antioxidant enzymes, resulting in the negative regulation of ROS homeostasis and CBC resistance in sweet orange plants. CsPrx53/CsSOD13 knockdown enhances ROS accumulation and CBC resistance. Overall, our results outline a regulatory pathway through which CsBZIP40 transcriptionally represses CsWRKY43-CsPrx53/CsSOD13 cascade-mediated ROS scavenging in a manner conducive to CBC resistance. These mechanisms underscore the potential importance of CsBZIP40, CsWRKY43, CsPrx53, and CsSOD13, providing promising strategies for the prevention of CBC.

A Lipid Droplet-Specific NIR Fluorescent Probe with a Large Stokes Shift for In Vivo Visualization of Polarity in Contrast-Induced Acute Kidney Injury.

The research on lipid droplets (LDs) has attracted great attention in the field of biomedical science in recent years. LD malfunction is found to be associated with the development of acute kidney injury (AKI). To monitor this biological process and explain related pathological behavior, the development of excellent LD fluorescent probes with a polarity-sensitive character would provide a desirable strategy. Herein, we designed a new polarity-susceptible fluorescent probe named LD-B with LD targetability, which exhibits very weak fluorescence in highly polar solvents based on the twisted intramolecular charge transfer effect but enhanced fluorescence in low polar environments, enabling us to visualize polarity alteration. The probe LD-B also possesses the merits of intense near-infrared (NIR) emission, good photostability, large Stokes shift, low toxicity, faster metabolic rate, and wash-free ability; thereby, it would contribute to efficient LD fluorescence visualization application. Using LD-B via confocal laser scanning fluorescence imaging and a small-animal imaging system in vivo, we first manifested a prominent rise of LD polarity in contrast-induced AKI (CI-AKI), not only at the cellular level but also in animals in vivo. Furthermore, the in vivo studies suggest that LD-B could accumulate in the kidney. In addition, the normal cell lines (including kidney cells) exhibiting a greater polarity of LDs than the cancer cells have been demonstrated systemically. Altogether, our work presents an effective approach for the medical diagnosis of LDs related to CI-AKI and identification of potential therapeutic markers.

The implication of chromosomal abnormalities in the surgical outcomes of Chinese pediatric patients with congenital heart disease.

Background: Copy 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. Methods: CNVs 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. Results: We 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 < 0.001). Duration of cardiopulmonary bypass and aortic cross clamp procedures were significantly longer in CHD cases with ppCNVs (all P < 0.05), while no group differences were identified for complications of surgery and one-month mortality after surgery. The detection rate of ppCNVs in the atrioventricular septal defect (AVSD) subgroup was significantly higher than that in other subgroups (23.10% vs. 9.70%, P = 0.002). Conclusions: CNV burden is an important contributor to Chinese children with CHD. Our study demonstrated the robustness and diagnostic efficiency of HLPA method in the genetic screening of CNVs in CHD patients.

CsAP2-09 confers resistance against citrus bacterial canker by regulating CsGH3.1L-mediated phytohormone biosynthesis.

Citrus bacterial canker (CBC) is a serious bacterial disease affecting citrus plantations and the citrus industry all over the world. We have previously shown that an apetala 2/ethylene response factor in Citrus sinensis, CsAP2-09, positively regulated resistance to CBC, although the regulatory mechanisms remained undetermined. Here, we demonstrated that CsAP2-09 positively and sustainably controlled resistance to CBC in three-year transgenic plants. CsAP2-09 was found to be a transcriptional activator, and qRT-PCR and dual luciferase assays showed that it controlled the expression CsGH3.1L. CsAP2-09 bound directly to the promotor of CsGH3.1L, shown by yeast one-hybrid assay, with the binding site confirmed by electrophoretic mobility shift assay. Biochemical assays showed that CsAP2-09 negatively regulated the biosynthesis of indole acetic acid (IAA) and positively regulated that of salicylic acid (SA) and ethylene, verified with transient overexpression of CsGH3.1L. The combination of these results with those of previous reports indicated that SA, ethylene, and IAA can directly regulate CBC resistance. Overall, we revealed a pathway whereby CsAP2-09 conferred CBC resistance by direct binding to the CsGH3.1L promoter, activating its expression and modulating IAA, SA, and ethylene biosynthesis. Our study indicates the potential value of manipulating CsAP2-09 and CsGH3.1L in the breeding of CBC-resistant citrus.

Pyramiding the antimicrobial PR1aCB and AATCB genes in 'Tarocco' blood orange (Citrus sinensis Osbeck) to enhance citrus canker resistance.

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is a major bacterial disease responsible for substantial economic losses in citrus-producing areas. To breed transgenic citrus plants with enhanced resistance to citrus canker, two antimicrobial peptide genes, PR1aCB and AATCB, were incorporated into 'Tarocco' blood orange (Citrus sinensis Osbeck) plants via co-transformation and sequential re-transformation. The presence of PR1aCB and AATCB in double transgenic plants was confirmed by PCR. The expression of PR1aCB and AATCB in double transformants was demonstrated by quantitative real-time PCR. An in vivo disease resistance assay involving the injection of Xcc revealed that the double transformants were more resistant to citrus canker than the single gene transformants and wild-type plants. An analysis of the bacterial population indicated that the enhanced citrus canker resistance of the double transformants was due to inhibited Xcc growth. These results proved that the pyramiding of multiple genes is a more effective strategy for increasing the disease resistance of transgenic citrus plants than single gene transformations.

Overexpression of Salicylic Acid Carboxyl Methyltransferase (CsSAMT1) Enhances Tolerance to Huanglongbing Disease in Wanjincheng Orange (Citrus sinensis (L.) Osbeck).

Citrus Huanglongbing (HLB) disease or citrus greening is caused by Candidatus Liberibacter asiaticus (Las) and is the most devastating disease in the global citrus industry. Salicylic acid (SA) plays a central role in regulating plant defenses against pathogenic attack. SA methyltransferase (SAMT) modulates SA homeostasis by converting SA to methyl salicylate (MeSA). Here, we report on the functions of the citrus SAMT (CsSAMT1) gene from HLB-susceptible Wanjincheng orange (Citrus sinensis (L.) Osbeck) in plant defenses against Las infection. The CsSAMT1 cDNA was expressed in yeast. Using in vitro enzyme assays, yeast expressing CsSAMT1 was confirmed to specifically catalyze the formation of MeSA using SA as a substrate. Transgenic Wanjincheng orange plants overexpressing CsSAMT1 had significantly increased levels of SA and MeSA compared to wild-type controls. HLB resistance was evaluated for two years and showed that transgenic plants displayed significantly alleviated symptoms including a lack of chlorosis, low bacterial counts, reduced hyperplasia of the phloem cells, and lower levels of starch and callose compared to wild-type plants. These data confirmed that CsSAMT1 overexpression confers an enhanced tolerance to Las in citrus fruits. RNA-seq analysis revealed that CsSAMT1 overexpression significantly upregulated the citrus defense response by enhancing the transcription of disease resistance genes. This study provides insight for improving host resistance to HLB by manipulation of SA signaling in citrus fruits.

Transcription factor WRKY22 regulates canker susceptibility in sweet orange (Citrus sinensis Osbeck) by enhancing cell enlargement and CsLOB1 expression.

Pathological hypertrophy (cell enlargement) plays an important role in the development of citrus canker, but its regulators are largely unknown. Although WRKY22 is known to be involved in pathogen-triggered immunity and positively regulates resistance to bacterial pathogens in Arabidopsis, rice and pepper, the CRISPR/Cas9-mediated partial knockout of CsWRKY22 improves resistance to Xanthomonas citri subsp. citri (Xcc) in Wanjincheng orange (Citrus sinensis Osbeck). Here, we demonstrate that CsWRKY22 is a nucleus-localized transcriptional activator. CsWRKY22-overexpressing plants exhibited dwarf phenotypes that had wrinkled and thickened leaves and were more sensitive to Xcc, whereas CsWRKY22-silenced plants showed no visible phenotype changes and were more resistant to Xcc. Microscopic observations revealed that the overexpression of CsWRKY22 increased cell size in the spongy mesophyll. Transcriptome analysis showed that cell growth-related pathways, such as the auxin and brassinosteroid hormonal signaling and cell wall organization and biogenesis pathways, were significantly upregulated upon CsWRKY22 overexpression. Interestingly, CsWRKY22 activated the expression of CsLOB1, which is a key gene regulating susceptibility to citrus canker. We further confirmed that CsWRKY22 bound directly to the W-boxes just upstream of the transcription start site of CsLOB1 in vivo and in vitro. We conclude that CsWRKY22 enhances susceptibility to citrus canker by promoting host hypertrophy and CsLOB1 expression. Thus, our study provides new insights into the mechanism regulating pathological hypertrophy and the function of WRKY22 in citrus.

CsLOB1 regulates susceptibility to citrus canker through promoting cell proliferation in citrus.

Citrus sinensis lateral organ boundary 1 (CsLOB1) was previously identified as a critical disease susceptibility gene for citrus bacterial canker, which is caused by Xanthomonas citri subsp. citri (Xcc). However, the molecular mechanisms of CsLOB1 in citrus response to Xcc are still elusive. Here, we constructed transgenic plants overexpressing and RNAi-silencing of CsLOB1 using the canker-disease susceptible 'wanjincheng' orange (C. sinensis Osbeck) as explants. CsLOB1-overexpressing plants exhibited dwarf phenotypes with smaller and thicker leaf, increased branches and adventitious buds clustered on stems. These phenotypes were followed by a process of pustule- and canker-like development that exhibited enhanced cell proliferation. Pectin depolymerization and expansin accumulation were enhanced by CsLOB1 overexpression, while cellulose and hemicellulose synthesis were increased by CsLOB1 silence. Whilst overexpression of CsLOB1 increased susceptibility, RNAi-silencing of CsLOB1 enhanced resistance to canker disease without impairing pathogen entry. Transcriptome analysis revealed that CsLOB1 positively regulated cell wall degradation and modification processes, cytokinin metabolism, and cell division. Additionally, 565 CsLOB1-targeted genes were identified in chromatin immunoprecipitation-sequencing (ChIP-seq) experiments. Motif discovery analysis revealed that the most highly overrepresented binding sites had a conserved 6-bp 'GCGGCG' consensus DNA motif. RNA-seq and ChIP-seq data suggested that CsLOB1 directly activates the expression of four genes involved in cell wall remodeling, and three genes that participate in cytokinin and brassinosteroid hormone pathways. Our findings indicate that CsLOB1 promotes cell proliferation by mechanisms depending on cell wall remodeling and phytohormone signaling, which may be critical to citrus canker development and bacterial growth in citrus.