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SUMMARY: 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. [ABSTRACT FROM AUTHOR]

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SUMMARY: Citrus production is severely threatened by the devastating Huanglongbing (HLB) disease globally. By studying and analyzing the defensive behaviors of an HLB‐tolerant citrus cultivar 'Shatangju', we discovered that citrus can sense Candidatus Liberibacter asiaticus (CLas) infection and induce immune responses against HLB, which can be further strengthened by both endogenously produced and exogenously applied methyl salicylate (MeSA). This immune circuit is turned on by an miR2977‐SAMT (encoding a citrus Salicylate [SA] O‐methyltransferase) cascade, by which CLas infection leads to more in planta MeSA production and aerial emission. We provided both transgenic and multi‐year trail evidences that MeSA is an effective community immune signal. Ambient MeSA accumulation and foliage application can effectively induce defense gene expression and significantly boost citrus performance. We also found that miRNAs are battle fields between citrus and CLas, and about 30% of the differential gene expression upon CLas infection are regulated by miRNAs. Furthermore, CLas hijacks host key processes by manipulating key citrus miRNAs, and citrus employs miRNAs that coordinately regulate defense‐related genes. Based on our results, we proposed that miRNAs and associated components are key targets for engineering or breeding resistant citrus varieties. We anticipate that MeSA‐based management, either induced expression or external application, would be a promising tool for HLB control. Significance Statement: The Great Wall not only served as a defensive barrier to ancient China, but also an intelligence transmission system. Foreign invasion alert at remote borders was signaled by rising smoke on the beacon towers and was relayed by the neighboring towers along the Great Wall. Recently researchers found a 'Great Wall' signaling system in citrus. Huanglongbing (HLB) is a devastating citrus disease causing billions of dollar losses annually. Research shows that citrus can produce and emit more methyl salicylate (MeSA) after being infected by CLas (the associated agent with HLB). MeSA is a violate that can spread through citrus growing groves and 'alarm' the uninfected citrus for the emerging threatens. This research also points out that microRNA (miRNA) is a key immune regulator in citrus. Image by: Hongwei Zhao. Due to our limited understanding of CLas pathogenicity and citrus immunity, citrus HLB causes enormous global economic losses yearly. In this investigation, we demonstrated that citrus can mount effective immune responses upon CLas infection, which is characterized by emitted community immune signal (MeSA) and citrus miRNA‐coordinated massive gene reprogramming. These two discoveries are of particular importance in the citrus industry. We could develop MeSA‐based HLB management strategies that would prime uninfected trees in the grove. In the future, we could also breed or engineer tolerant or even resistant citrus varieties and rootstocks relying on modifying critical citrus miRNAs. Our findings revealed not only citrus defensive tool kits against the HLB but also the potential of utilizing these weapons practically. [ABSTRACT FROM AUTHOR]

Autophagy functions in plant host immunity responses to pathogen infection. The molecular mechanisms and functions used by the citrus Huanglongbing (HLB)-associated intracellular bacterium ' Candidatus Liberibacter asiaticus' (C Las) to manipulate autophagy are unknown. We identified a C Las effector, SDE4405 (CLIBASIA_04405), which contributes to HLB progression. 'Wanjincheng' orange (Citrus sinensi s) transgenic plants expressing SDE4405 promotes C Las proliferation and symptom expression via suppressing host immunity responses. SDE4405 interacts with the ATG8-family of proteins (ATG8s), and their interactions activate autophagy in Nicotiana benthamiana. The occurrence of autophagy is also significantly enhanced in SDE4405-transgenic citrus plants. Interrupting NbATG8s-SDE4405 interaction by silencing of NbATG8c reduces Pseudomonas syringae pv. tomato strain DC3000 ΔhopQ1-1 (Pst DC3000 ΔhopQ1-1) proliferation in N. benthamiana , and transient overexpression of CsATG8c and SDE4405 in citrus promotes Xanthomonas citri subsp. citri (Xcc) multiplication, suggesting that SDE4405-ATG8s interaction negatively regulates plant defense. These results demonstrate the role of the C Las effector protein in manipulating autophagy, and provide new molecular insights into the interaction between C Las and citrus hosts. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB), caused by " Candidatus liberibacter asiaticus" (Ca Las), is one of the most devastating diseases in citrus but its pathogenesis remains poorly understood. Here, we reported the role of the Ca LasSDE115 (CLIBASIA_05115) effector, encoded by Ca Las, during pathogen-host interactions. Bioinformatics analyses showed that Ca LasSDE115 was 100% conserved in all reported Ca Las strains but had sequence differences compared with orthologs from other " Candidatus liberibacter." Prediction of protein structures suggested that the crystal structure of Ca LasSDE115 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 Ca LasSDE115 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 Ca Las-infected Asian citrus psyllid (ACP) were much higher (∼45-fold) than those in Ca Las-infected Wanjincheng oranges, with the expression in symptomatic leaves being significantly higher than that in asymptomatic ones. Additionally, the overexpression of mSDE115 favored Ca Las 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 Ca LasSDE115 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. [ABSTRACT FROM AUTHOR]

Summary: 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. [ABSTRACT FROM AUTHOR]

Flower-specific promoters enable genetic manipulation of floral organs to improve crop yield and quality without affecting vegetative growth. However, the identification of strong tissue-specific promoters is a challenge. In addition, information on cis elements that is able to repress gene expression in vegetative tissues remains limited. Here, we report that fusing a 35S enhancer to the stamen- and carpel-specific NtAGIP1 promoter derived from the tobacco AGAMOUS second intron (AGI) can significantly increase the promoter activity. Interestingly, although the activity of the new promoter extends to sepals and pedicles, it does not cross the boundary of the reproductive organs. Serial deletion of the AGI and chromatin immunoprecipitation (ChIP) assay reveal a 100-bp fragment that contains a conserved GAGA factor binding motif contributes to the flower specificity by mediating histone H3 lysine 27 trimethylation (H3K27me3) modification of the promoter. Furthermore, this fragment shows significant suppressive effect on the activity of the 35S enhancer in vegetative tissues, consequently, resulting in a significant increase of the activity of 35S enhancer:AGI chimeric promoter without sacrifice of its specificity in inflorescence. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 Cs LOB1 promoter in citrus. Wanjincheng orange ( Citrus sinensis Osbeck) harbours at least three copies of the Cs LOB1G allele and one copy of the Cs LOB1− allele. The promoter of both alleles contains the effector binding element ( EBEPthA4), which is recognized by the main effector PthA4 of Xcc to activate Cs LOB1 expression to promote citrus canker development. Five pCas9/Cs LOB1sg RNA constructs were designed to modify the EBEPthA4 of the Cs LOB1 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 EBEPthA4 modifications were identified from 38 mutant plants. Four mutation lines (S2-5, S2-6, S2-12 and S5-13), in which promoter editing disrupted Cs LOB1 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 Cs LOB1G alone was sufficient to enhance citrus canker resistance in Wanjincheng orange. Deletion of the entire EBEPthA4 sequence from both Cs LOB1 alleles conferred a high degree of resistance to citrus canker. The results demonstrate that CRISPR/Cas9-mediated promoter editing of Cs LOB1 is an efficient strategy for generation of canker-resistant citrus cultivars. [ABSTRACT FROM AUTHOR]

Marker-free transgenic plants alleviate concerns regarding the biosafety of genetically modified organisms and promote their commercialization. In this study, a transformation vector pLI35SAAT, harboring a Cre/ loxP-mediated recombination system combined with the isopentenyl transferase ( ipt) selectable marker gene and an anti-bacterial peptide gene AATCB, was used to produce marker-free transgenic 'Tarocco' blood orange ( Citrus sinensis Osbeck) with enhanced resistance to citrus canker. Using ipt positive selection, a transformation efficiency of 21.4 % was achieved. When the DNA between two loxP sites was excised, phenotypically normal shoots gradually appeared from 74.8 % of the transgenic ipt shoots. Their marker-free transgenic nature was confirmed using PCR and sequencing analyses. In vitro evaluations of citrus canker disease resistance revealed that marker-free transgenic plants exhibited an enhanced resistance to Xanthomonas axonopodis pv. citri. The marker-free transgenic plants appeared phenotypically normal under greenhouse conditions. Thus, marker-free transgenic citrus plants with targeted traits can be efficiently produced using a Cre/ loxP-mediated recombination system combined with ipt positive selection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) and citrus canker, arising from Candidatus Liberibacter asiaticus ( Ca Las) 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 , CsSABP2-1 V18A , 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 CsSABP2-1 V18A has the strongest activity. Their activities were inhibited by SA except for CsSABP2-1 V18A . 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 CsSABP2-1 V18A had the strongest action on SA. Resistance evaluation demonstrated that only CsSABP2-1 V18A 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Dong, Chen, Shang, Du, Mo, Pang, Zheng, Xu, Lei, He and Zou.)

Huanglongbing (HLB) primarily caused by Candidatus Liberibacter asiaticus ( C Las) 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 C Las 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 C Las-infected leaves (correlation coefficient of 0.77, P  ≤ 0.05). Knockdown of CsCalS11 resulted in a reduction of callose deposition and starch accumulation in C Las-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 C Las 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 C Las infection. The results also revealed new function of the ABA signaling pathway in plants under biotic stress., Competing Interests: The authors have no conflict of interest to declare., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nanjing Agricultural University.)

The most damaging citrus diseases are Huanglongbing (HLB) and citrus canker, which are caused by Candidatus Liberibacter asiaticus ( Ca Las) and Xanthomonas citri pv. citri ( Xcc ), respectively. Endolysins from bacteriophages are a possible option for disease resistance in plant breeding. Here, we report improvement of citrus resistance to HLB and citrus canker using the LasLYS1 and LasLYS2 endolysins from Ca Las. LasLYS2 demonstrated bactericidal efficacy against several Rhizobiaceae bacteria and Xcc , according to inhibition zone analyses. The two genes, driven by a strong promoter from Cauliflower mosaic virus , 35S, were integrated into Carrizo citrange via Agrobacterium -mediated transformation. More than 2 years of greenhouse testing indicated that LasLYS2 provided substantial and long-lasting resistance to HLB, allowing transgenic plants to retain low Ca Las titers and no obvious symptoms while also clearing Ca Las from infected plants in the long term. LasLYS2 transgenic plants with improved HLB resistance also showed resistance to Xcc , indicating that LasLYS2 had dual resistance to HLB and citrus canker. A microbiome study of transgenic plants revealed that the endolysins repressed Xanthomonadaceae and Rhizobiaceae populations in roots while increasing Burkholderiaceae and Rhodanobacteraceae populations, which might boost the citrus defense response, according to transcriptome analysis. We also found that Lyz domain 2 is the key bactericidal motif of LasLYS1 and LasLYS2. Four endolysins with potential resistance to HLB and citrus canker were found based on the structures of LasLYS1 and LasLYS2. Overall, the work shed light on the mechanisms of resistance of Ca Las-derived endolysins, providing insights for designing endolysins to develop broad-spectrum disease resistance in citrus., Competing Interests: The authors declare that they have no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nanjing Agricultural University.)

Although emerging evidence indicates that bacteria extracellularly export many cytoplasmic proteins referred to as non-classically secreted proteins (ncSecPs) for their own benefit, the mechanisms and functional significance of the ncSecPs in extracellular milieu remain elusive. " Candidatus Liberibacter asiaticus" (CLas) is a fastidious Gram-negative bacterium that causes Huanglongbing (HLB), the most globally devastating citrus disease. In this study, using the SecretomeP program coupled with an Escherichia coli alkaline phosphatase assay, we identified 27 ncSecPs from the CLas genome. Further, we demonstrated that 10 of these exhibited significantly higher levels of gene expression in citrus than in psyllid hosts, and particularly suppressed hypersensitive response (HR)-based cell death and H 2 O 2 overaccumulation in Nicotiana benthamiana , indicating their opposing effects on early plant defenses. However, these proteins also dramatically enhanced the gene expression of pathogenesis-related 1 protein (PR-1), PR-2, and PR-5, essential components of plant defense mechanisms. Additional experiments disclosed that the increased expression of these PR genes, in particular PR-1 and PR-5 , could negatively regulate HR-based cell death development and H 2 O 2 accumulation. Remarkably, CLas infection clearly induced gene expression of PR-1, PR-2, and PR-5 in both HLB-tolerant and HLB-susceptible species of citrus plants. Taken together, we hypothesized that CLas has evolved an arsenal of ncSecPs that function cooperatively to overwhelm the early plant defenses by inducing host PR genes. IMPORTANCE In this study, we present a combined computational and experimental methodology that allows a rapid and efficient identification of the ncSecPs from bacteria, in particular the unculturable bacteria like CLas. Meanwhile, the study determined that a number of CLas ncSecPs suppressed HR-based cell death, and thus indicated a novel role for the bacterial ncSecPs in extracellular milieu. More importantly, these ncSecPs were found to suppress cell death presumably by utilizing host PR proteins. The data overall provide a novel clue to understand the CLas pathogenesis and also suggest a new way by which phytopathogens manipulate host cellular machinery to establish infection., (Copyright © 2021 American Society for Microbiology.)

CRISPR/Cas9 has been widely employed to edit genome in most of the organisms, including animal, plant, fungus, and microbe. Here we describe the modification of citrus gene CsLOB1 in transgenic citrus by Cas9/sgRNA, a two-component system derived from CRISPR-Cas9. Transgenic citrus plants can be created by Agrobacterium-mediated epicotyl transformation.