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Pustule formation is pivotal for the development of the Xanthomonas citri subsp. citri (Xcc)-induced citrus canker disease (CCD). Although our previous study demonstrated that the exogenous application of abscisic acid (ABA) facilitated pustule formation induced by Xcc, the precise mechanism remains elusive. The 9-cis-epoxycarotenoid dioxygenase (NCED) is a crucial enzyme in ABA biosynthesis. This study explored the role of citrus CsNCED1-1 in CCD resistance through overexpression and RNA interference of CsNCED1-1 in Wanjincheng orange (Citrus sinensis). Our findings indicated that CsNCED1-1 negatively modulated CCD resistance by fostering ABA accumulation, concomitant with an increase in jasmonic acid (JA) and a decrease in salicylic acid (SA). Plants overexpressing CsNCED1-1 displayed shortened leaves with smaller and denser stomata along with irregular and increased palisade cells. CsLOB1 is a known susceptibility gene for CCD, and CsbZIP40 positively influences resistance to this disease. We further confirmed that CsLOB1 promoted and CsbZIP40 suppressed the transcription of CsNCED1-1 by directly binding to the CsNCED1-1 promoter. Notably, CsbZIP40 and CsLOB1 showed a competitive relationship in the regulation of CsNCED1-1 expression, with CsbZIP40 exhibiting greater competitiveness. Overall, our findings highlight that CsNCED1-1 promotes susceptibility to citrus canker by disrupting JA- and SA-mediated defense mechanisms and triggering the proliferation and remodeling of palisade cells, thereby facilitating pathogen colonization and pustule formation. This study offers novel insights into the regulatory mechanisms underlying citrus canker resistance and the role of CsNCED1-1 in citrus., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

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., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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., Competing Interests: Declaration of competing interest The authors declare no conflict of interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

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.

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., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

The present study was designed to serve as a comprehensive analysis of Citrus sinensis ( C. sinensis ) pectin acetylesterases (CsPAEs), and to assess the roles of these PAEs involved in the development of citrus bacterial canker (CBC) caused by Xanthomonas citri subsp. citri ( Xcc ) infection. A total of six CsPAEs were identified in the genome of C. sinensis , with these genes being unevenly distributed across chromosomes 3, 6, and 9, and the unassembled scaffolds. A subset of CsPAEs were found to be involved in responses to Xcc infection. In particular, CsPAE2 was identified to be associated with such infections, as it was upregulated in CBC-susceptible variety Wanjincheng and inversely in CBC-resistant variety Calamondin. Transgenic citrus plants overexpressing CsPAE2 were found to be more susceptible to CBC, whereas the silencing of this gene was sufficient to confer CBC resistance. Together, these findings provide evolutionary insights into and functional information about the CsPAE family. This study also suggests that CsPAE2 is a potential candidate gene that negatively contributes to bacterial canker disease and can be used to breed CBC-resistant citrus plants.

Class III peroxidase (CIII prx) is a plant-specific multigene family that regulates the physiological and stress responses. This research aimed to exhaustively annotate and analyse the CIII prx family in sweet orange and to explore the regulated expression profiles by Xanthomonas citri subsp. citri ( Xcc ) and plant hormones. We further assessed the relationship between CIII prxs and citrus bacterial canker. The phylogeny, gene structure, conserved motifs, gene duplications and microsynteny of the CIII prx family were analysed. Expression profiles of specific CsPrxs induced by Xanthomonas citri subsp. citri and plant hormones were detected by quantitative reverse transcription-polymerase chain reaction. Subcellular localization was analysed through transient expression assessments. A total of 72 CIII prx members were identified from the genomes of sweet orange. In all chromosomes of sweet orange, the CsPrxs could be detected except in chromosome 8. In addition, three segmental duplications, four tandem duplications and 11 whole-genome duplications occurred among the CsPrxs, contributing to the family size expansion. From the K a / K s ratios, 15 of 18 duplicated CsPrxs pairs have experienced purifying selection process. A total of 15 conserved motifs were detected in CsPrxs, four of which were detected in all complete CsPrxs. A total of 12 expressed genes were identified from the EST database. The expression trends of 12 CsPrxs were differently expressed at different stages of infection by Xcc , five of which were potential candidate genes involved in Xcc resistance. These genes could be induced by salicylic acidand methyl jasmonate, and were extracellular proteins. These results further support our understanding of CIII prxs in citrus, particularly incitrus bacterial canker studies.

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., Competing Interests: The authors have declared that no competing interests exist.

Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is severely damaging to the global citrus industry. Targeted editing of host disease-susceptibility genes represents an interesting and potentially durable alternative in plant breeding for resistance. Here, we report improvement of citrus canker resistance through CRISPR/Cas9-targeted modification of the susceptibility gene CsLOB1 promoter in citrus. Wanjincheng orange (Citrus sinensis Osbeck) harbours at least three copies of the CsLOB1 G allele and one copy of the CsLOB1 - allele. The promoter of both alleles contains the effector binding element (EBE P thA4 ), which is recognized by the main effector PthA4 of Xcc to activate CsLOB1 expression to promote citrus canker development. Five pCas9/CsLOB1sgRNA constructs were designed to modify the EBE P thA4 of the CsLOB1 promoter in Wanjincheng orange. Among these constructs, mutation rates were 11.5%-64.7%. Homozygous mutants were generated directly from citrus explants. Sixteen lines that harboured EBE P thA4 modifications were identified from 38 mutant plants. Four mutation lines (S2-5, S2-6, S2-12 and S5-13), in which promoter editing disrupted CsLOB1 induction in response to Xcc infection, showed enhanced resistance to citrus canker compared with the wild type. No canker symptoms were observed in the S2-6 and S5-13 lines. Promoter editing of CsLOB1 G alone was sufficient to enhance citrus canker resistance in Wanjincheng orange. Deletion of the entire EBE P thA4 sequence from both CsLOB1 alleles conferred a high degree of resistance to citrus canker. The results demonstrate that CRISPR/Cas9-mediated promoter editing of CsLOB1 is an efficient strategy for generation of canker-resistant citrus cultivars., (© 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Citrus bacterial canker (CBC) is a disease resulting from Xanthomonas citri subsp. citri (Xcc) infection and poses a grave threat to citrus production worldwide. Wall-associated receptor-like kinases (WAKLs) are proteins with a central role in resisting a range of fungal and bacterial diseases. The roles of WAKLs in the context of CBC resistance, however, remain unclear. Here, we explored the role of CsWAKL08, which confers resistance to CBC, and we additionally analyzed the molecular mechanisms of CsWAKL08-mediated CBC resistance. Based on systematic annotation and induced expression analysis of the CsWAKL family in Citrus sinensis, CsWAKL08 was identified as a candidate that can be upregulated by Xcc infection in the CBC-resistant variety. CsWAKL08 can also be induced by the phytohormones salicylic acid (SA) and methyl jasmonic acid (MeJA) and spans the plasma membrane. Overexpression of CsWAKL08 resulted in strong CBC resistance in transgenic sweet oranges, whereas silencing of CsWAKL08 resulted in susceptibility to CBC. The peroxidase (POD) and superoxide dismutase (SOD) activities were significantly enhanced in the CsWAKL08-overexpressing plants compared to the control plants, thereby mediating reactive oxygen species (ROS) homeostasis in the transgenic plants. Moreover, the JA levels and the expression of JA biosynthesis and JA responsive genes were substantially elevated in the CsWAKL08 overexpression plants relative to the controls upon Xcc infection. Based on these findings, we conclude that the wall-associated receptor-like kinase CsWAKL08 positively regulates CBC resistance through a mechanism involving ROS control and JA signaling. These results further highlight the importance of this kinase family in plant pathogen resistance. [ABSTRACT FROM AUTHOR]