Citrus Huanglongbing Detection Based on Multi-Modal Feature Fusion Learning.
Frontiers in plant science
Citrus Huanglongbing (HLB), also named citrus greening disease, occurs worldwide and is known as a citrus cancer without an effective treatment. The symptoms of HLB are similar to those of nutritional deficiency or other disease. The methods based on single-source information, such as RGB images or hyperspectral data, are not able to achieve great detection performance. In this study, a multi-modal feature fusion network, combining a RGB image network and hyperspectral band extraction network, was proposed to recognize HLB from four categories (HLB, suspected HLB, Zn-deficient, and healthy). Three contributions including a dimension-reduction scheme for hyperspectral data based on a soft attention mechanism, a feature fusion proposal based on a bilinear fusion method, and auxiliary classifiers to extract more useful information are introduced in this manuscript. The multi-modal feature fusion network can effectively classify the above four types of citrus leaves and is better than single-modal classifiers. In experiments, the highest accuracy of multi-modal network recognition was 97.89% when the amount of data was not very abundant (1,325 images of the four aforementioned types and 1,325 pieces of hyperspectral data), while the single-modal network with RGB images only achieved 87.98% recognition and the single-modal network using hyperspectral information only 89%. Results show that the proposed multi-modal network implementing the concept of multi-source information fusion provides a better way to detect citrus HLB and citrus deficiency.
10.3389/fpls.2021.809506
' Liberibacter Asiaticus' SDE1 Effector Induces Huanglongbing Chlorosis by Downregulating Host Gene.
International journal of molecular sciences
' Liberibacter asiaticus' (CLas) is the pathogenic bacterium that causes the disease Huanglongbing (HLB) in citrus and some model plants, such as . After infection, CLas releases a set of effectors to modulate host responses. One of these critical effectors is Sec-delivered effector 1 (SDE1), which induces chlorosis and cell death in . In this study, we revealed the DEAD-box RNA helicase (DDX3) interacts with SDE1. Gene silencing study revealed that knockdown of the gene triggers leaf chlorosis, mimicking the primary symptom of CLas infection in . The interactions between SDE1 and NbDDX3 were localized in the cell membrane. Overexpression of resulted in suppression of gene expression in , which suggests a critical role of in modulating expression. Furthermore, we verified the interaction of SDE1 with citrus DDX3 (CsDDX3), and demonstrated that the expression of the gene was significantly reduced in HLB-affected yellowing and mottled leaves of citrus. Thus, we provide molecular evidence that the downregulation of the host gene is a crucial mechanism of leaf chlorosis in HLB-affected plants. The identification of CsDDX3 as a critical target of SDE1 and its association with HLB symptom development indicates that the gene is an important target for gene editing, to interrupt the interaction between DDX3 and SDE1, and therefore interfere host susceptibility.
10.3390/ijms21217996
Annotation of genes in , the vector for Huanglongbing disease.
GigaByte (Hong Kong, China)
Huanglongbing (HLB), also known as citrus greening disease, is caused by the bacterium Liberibacter asiaticus (Las). It is a serious threat to global citrus production. This bacterium is transmitted by the Asian citrus psyllid, (Hemiptera). There are no effective treatments for Las. Therefore, one strategy is to manage the psyllid population. Manual annotation of the genome can identify and characterize gene families that could be novel targets for psyllid control. The gene family is an excellent target because genes, which have roles in melanization, are linked to development and immunity. Combined analysis of the genome with RNA-seq datasets, sequence homology, and phylogenetic trees were used to identify and annotate nine genes in the genome. Manual curation of genes in provided in-depth analysis of the family among hemipteran insects and provides new targets for molecular control of this psyllid pest. Manual annotation was done as part of a collaborative Citrus Greening community annotation project.
10.46471/gigabyte.20
Distribution and dynamic changes of Huanglongbing pathogen in its insect vector .
Frontiers in cellular and infection microbiology
The Asian citrus psyllid (ACP) Kuwayama is the leading vector of Liberibacter asiaticus (Las), the causative agent of citrus Huanglongbing (HLB) disease. The distribution and dynamics of Las within ACP are critical to understanding how the transmission, spread and infection of Las occurs within its host vector in nature. In this study, the distribution and titer changes of Las in various tissues of ACP 5 instar nymphs and adults were examined by (FISH) and real-time quantitative PCR (qPCR) techniques. Results demonstrated that 100% of ACP 5 instar nymphs and adults were infected with Las following feeding on infected plants, and that Las had widespread distribution in most of the tissues of ACP. The titers of Las within the midgut, salivary glands and hemolymph tissues were the highest in both 5 instar nymphs and adults. When compared with adults, the titers of Las in these three tissues of 5 instar nymphs were significantly higher, while in the mycetome, ovary and testes they were significantly lower than those of adults. FISH visualization further confirmed these findings. Dynamic analysis of Las demonstrated that it was present across all the developmental ages of ACP adults. There was a discernible upward trend in the presence of Las with advancing age in most tissues of ACP adults, including the midgut, hemolymph, salivary glands, foot, head, cuticula and muscle. Our findings have significant implications for the comprehensive understanding of the transmission, dissemination and infestation of Las, which is of much importance for developing novel strategies to halt the spread of Las, and therefore contribute to the efficient prevention and control of HLB.
10.3389/fcimb.2024.1408362
Highly Sensitive and Rapid Detection of Citrus Huanglongbing Pathogen (' Liberibacter asiaticus') Using Cas12a-Based Methods.
Wheatley Matthew S,Duan Yong-Ping,Yang Yinong
Phytopathology
Citrus huanglongbing (HLB) or greening is one of the most devastating diseases of citrus worldwide. Sensitive detection of its causal agent, ' Liberibacter asiaticus' (CLas), is critical for early diagnosis and successful management of HLB. However, current nucleic acid-based detection methods are often insufficient for the early detection of CLas from asymptomatic tissue and unsuitable for high-throughput and field-deployable diagnosis of HLB. Here we report the development of the Cas12a-based DNA endonuclease-targeted CRISPR trans reporter (DETECTR) assay for highly specific and sensitive detection of CLas nucleic acids from infected samples. The DETECTR assay, which targets the five-copy gene specific to CLas, couples isothermal amplification with Cas12a transcleavage of a fluorescent reporter oligonucleotide and enables detection of CLas nucleic acids at the attomolar level. The DETECTR assay was capable of specifically detecting the presence of CLas across different infected citrus, periwinkle, and psyllid samples and shown to be compatible with lateral flow assay technology for potential field-deployable diagnosis. The improvements in detection sensitivity and flexibility of the DETECTR technology position the assay as a potentially suitable tool for early detection of CLas in infected regions.
10.1094/PHYTO-09-20-0443-R
The Upregulated Expression of the Citrus Gene in HLB Diseased Citrus Aids Liberibacter Asiaticus Infection.
International journal of molecular sciences
The citrus industry has been threatened by Huanglongbing (HLB) for over a century. Here, an HLB-induced Arabidopsis () homologous gene was cloned from , and its characteristics and function were analyzed to determine its role during citrus- Liberibacter asiaticus (Las) interactions. Quantitative real-time PCR showed that was expressed in roots, stems, leaves and flowers, with the greatest expression level in leaves. Its expression was suppressed by gibberellic acid, indole-3-acetic acid, salicylic acid and jasmonic acid treatments, but was induced by abscisic acid and salt treatments, as well as wounding. The transient expression of a RIN4-GFP showed that RIN4 was localized in the cell membrane. -overexpressing transgenic cv. 'Shatianyou' plants were obtained, and some transgenic plants showed greater sensitivity to Las infection and earlier HLB symptoms appearance than non-transgenic controls. Results obtained in this study indicated that the upregulated expression of in HLB diseased citrus may aid Las infection.
10.3390/ijms23136971
Uncovering Interactions between Plant Metabolism and Plant-Associated Bacteria in Huanglongbing-Affected Citrus Cultivars Using Multiomics Analysis and Machine Learning.
Journal of agricultural and food chemistry
Huanglongbing (HLB) is a highly destructive disease that inflicts significant economic losses on the citrus industry worldwide but with no cure available. However, microbiomes formulated by citrus plants may serve as disease antagonists, increasing the level of HLB tolerance. This study established an integrated analysis of untargeted metabolomics and microbiomics data for different citrus cultivars, providing critical insights into the interactions between plant metabolism and plant-associated bacteria in the development of HLB. Machine learning models were applied to screen important metabolites and bacteria in multiple citrus materials, and the selected metabolites were then analyzed to identify essential pathways enriched in the plant and to correlate with the selected bacteria. Results demonstrated that the regulation of plant pathways, especially ABC transporters and ubiquinone and other terpene-ubiquinone biosynthesis pathways, could affect the microbial community structure, indicating potential solutions for controlling HLB by modulating bacteria in citrus plants or breeding tolerant citrus cultivars.
10.1021/acs.jafc.3c04460
Control of Citrus Huanglongbing via Trunk Injection of Plant Defense Activators and Antibiotics.
Hu J,Jiang J,Wang N
Phytopathology
Citrus huanglongbing (HLB) or greening is a devastating disease of citrus worldwide and no effective control measure is currently available. Plant defense activators environmentally friendly compounds capable of inducing resistance against many plant pathogens. Earlier studies showed that foliar spray of plant defense inducers could slow down HLB disease progress. In this study, eight plant defense activators and three antibiotics were evaluated in three field trials for their effect to control HLB by trunk injection of young and mature sweet orange trees. Results showed that four trunk injections of several activators, including salicylic acid, oxalic acid, acibenzolar-S-methyl, and potassium phosphate, provided significant control of HLB by suppressing 'Candidatus Liberibacter asiaticus' titer and disease progress. Trunk injection of penicillin, streptomycin, and oxytetracycline hydrochloride resulted in excellent control of HLB. In general, antibiotics were more effective in reduction of 'Ca. L. asiaticus' titer and HLB symptom expressions than plant defense activators. These treatments also resulted in increased yield and better fruit quality. Injection of both salicylic acid and acibenzolar-S-methyl led to significant induction of pathogenesis-related (PR) genes PR-1 and PR-2 genes. Meanwhile, injection of either potassium phosphate or oxalic acid resulted in significant induction of PR-2 or PR-15 gene expression, respectively. These results suggested that HLB diseased trees remained inducible for systemic acquired resistance under field conditions. In summary, this study presents information regarding controlling HLB via trunk injection of plant defense activators and antibiotics, which helps citrus growers in decision making regarding developing an effective HLB management program.
10.1094/PHYTO-05-17-0175-R
Temporal and spatial detection of Candidatus Liberibacter asiaticus putative effector transcripts during interaction with Huanglongbing-susceptible, -tolerant, and -resistant citrus hosts.
Shi Qingchun,Pitino Marco,Zhang Shujian,Krystel Joseph,Cano Liliana M,Shatters Robert G,Hall David G,Stover Ed
BMC plant biology
BACKGROUND:Citrus Huanglongbing (HLB) is a bacterial disease with high economic significance. The associated agent Candidatus Liberibacter asiaticus is a fastidious, phloem-limited, intracellular bacterium that is transmitted by an insect vector the Asian citrus psyllid (ACP). The genome of Ca. L. asiaticus contains protein secretion machinery that suggests host cell modulation capacity of this bacterium. RESULTS:A total of 28 candidate effectors, an important class of secreted proteins, were predicted from the Ca. L. asiaticus genome. Sequence specific primers were designed for reverse transcription (RT) and quantitative PCR (qPCR), and expression was validated for 20 of the effector candidates in infected citrus with multiple genetic background. Using detached leaf inoculation, the mRNA of effectors was detected from 6 h to 7 days post ACP exposure. It was observed that higher bacterial titers were associated with a larger number of effectors showing amplification across all samples. The effectors' expression were compared in citrus hosts with various levels of HLB tolerance, including susceptible Duncan grapefruit and Washington navel orange, tolerant citron and Cleopatra mandarin, and resistant Pomeroy trifoliate and Carrizo citrange. Across all genotypes relatively high expression was observed for CLIBASIA_03695, CLIBASIA_00460, CLIBASIA_00420, CLIBASIA_04580, CLIBASIA_05320, CLIBASIA_04425, CLIBASIA_00525 and CLIBASIA_05315 in either a host-specific or -nonspecific manners. The two genotypes in each HLB-response group also show effector-expression profiles that seem to be different. In a companion study, the expression of effectors was compared between leaves and roots of own-rooted citrus that had been Ca. L. asiaticus-infected for more than a year. Results indicated relatively high expression of CLIBASIA_03875, CLIBASIA_04800 and CLIBASIA_05640 in all leaf and some root tissues of citron, Duncan and Cleopatra. CONCLUSION:This temporal and spatial expression analysis of Ca. L. asiaticus effectors identified candidates possibly critical for early bacterial colonization, host tolerance suppression and long-term survival which are all worthy of further investigation.
10.1186/s12870-019-1703-4
Three new discovery effector proteins from Candidatus Liberibacter asiaticus psy62 inhibit plant defense through interaction with AtCAT3 and AtGAPA.
Plant cell reports
KEY MESSAGE:We identify three SDEs that inhibiting host defence from Candidatus Liberibacter asiaticus psy62, which is an important supplement to the pathogenesis of HLB. Candidatus Liberibacter asiaticus (CLas) is the main pathogen of citrus Huanglongbing (HLB). 38 new possible sec-dependent effectors (SDEs) of CLas psy62 were predicted by updated predictor SignalP 5.0, which 12 new SDEs were found using alkaline phosphate assay. Among them, SDE4310, SDE4435 and SDE4955 inhibited hypersensitivity reactions (HR) in Arabidopsis thaliana (Arabidopsis, At) and Nicotiana benthamiana leaves induced by pathogens, which lead to a decrease in cell death and reactive oxygen species (ROS) accumulation. And the expression levels of SDE4310, SDE4435, and SDE4955 genes elevated significantly in mild symptom citrus leaves. When SDE4310, SDE4435 and SDE4955 were overexpressed in Arabidopsis, HR pathway key genes pathogenesis-related 2 (PR2), PR5, nonexpressor of pathogenesis-related 1 (NPR1) and isochorismate synthase 1 (ICS1) expression significantly decreased and the growth of pathogen was greatly increased relative to control with Pst DC3000/AvrRps4 treatment. Our findings also indicated that SDE4310, SDE4435 and SDE4955 interacted with AtCAT3 (catalase 3) and AtGAPA (glyceraldehyde-3-phosphate dehydrogenase A). In conclusion, our results suggest that SDE4310, SDE4435 and SDE4955 are CLas psy62 effector proteins that may have redundant functions. They inhibit ROS burst and cell death by interacting with AtCAT3 and AtGAPA to negatively regulate host defense.
10.1007/s00299-024-03220-z
A prophage-encoded effector from "Candidatus Liberibacter asiaticus" targets ASCORBATE PEROXIDASE6 in citrus to facilitate bacterial infection.
Molecular plant pathology
Citrus huanglongbing (HLB), associated with the unculturable phloem-limited bacterium "Candidatus Liberibacter asiaticus" (CLas), is the most devastating disease in the citrus industry worldwide. However, the pathogenicity of CLas remains poorly understood. In this study, we show that AGH17488, a secreted protein encoded by the prophage region of the CLas genome, suppresses plant immunity via targeting the host ASCORBATE PEROXIDASE6 (APX6) protein in Nicotiana benthamiana and Citrus sinensis. The transient expression of AGH17488 reduced the chloroplast localization of APX6 and its enzyme activity, inhibited the accumulation of reactive oxygen species (H O and O ) and the lipid oxidation endproduct malondialdehyde in plants, and promoted the proliferation of Pseudomonas syringae pv. tomato DC3000 and Xanthomonas citri subsp. citri. This study reveals a novel mechanism underlying how CLas uses a prophage-encoded effector, AGH17488, to target a reactive oxygen species accumulation-related gene, APX6, in the host to facilitate its infection.
10.1111/mpp.13296
Identifying Host Molecular Features Strongly Linked With Responses to Huanglongbing Disease in Citrus Leaves.
Balan Bipin,Ibáñez Ana M,Dandekar Abhaya M,Caruso Tiziano,Martinelli Federico
Frontiers in plant science
A bioinformatic analysis of previously published RNA-Seq studies on Huanglongbing (HLB) response and tolerance in leaf tissues was performed. The aim was to identify genes commonly modulated between studies and genes, pathways and gene set categories strongly associated with this devastating Citrus disease. Bioinformatic analysis of expression data of four datasets present in NCBI provided 46-68 million reads with an alignment percentage of 72.95-86.76%. Only 16 HLB-regulated genes were commonly identified between the three leaf datasets. Among them were key genes encoding proteins involved in cell wall modification such as CESA8, pectinesterase, expansin8, expansin beta 3.1, and a pectate lyase. Fourteen HLB-regulated genes were in common between all four datasets. Gene set enrichment analysis showed some different gene categories affected by HLB disease. Although sucrose and starch metabolism was highly linked with disease symptoms, different genes were significantly regulated depending on leaf growth and infection stages and experimental conditions. Histone-related transcription factors were highly affected by HLB in the analyzed RNA-Seq datasets. HLB tolerance was linked with induction of proteins involved in detoxification. Protein-protein interaction (PPI) network analysis confirmed a possible role for heat shock proteins in curbing disease progression.
10.3389/fpls.2018.00277
Modeling Study of the Effects of on the Transmission and Control of Citrus Huanglongbing.
Plants (Basel, Switzerland)
() is commonly found or intentionally planted in citrus orchards due to its ability to provide habitat and breeding grounds for the natural enemies of citrus pests. This study aims to expand from a switching Huanglongbing model by incorporating the effects of , vector preferences for settling, and pesticide application intervals on disease transmission. Additionally, we establish the basic reproduction number R0 and its calculation for a general switching compartmental epidemic model. Theoretical findings demonstrate that the basic reproduction number serves as a threshold parameter to characterize the dynamics of the models: if R0<1, the disease will disappear, whereas if R0>1, it will spread. Numerical results indicate that the recruitment rate of not only affects the spread speed of Huanglongbing but also leads to paradoxical effects. Specifically, in cases of high infection rates, a low recruitment rate of can result in a decrease, rather than an increase, in the basic reproduction number. Conversely, a high recruitment rate can accelerate the spread of Huanglongbing. Furthermore, we show how different vector bias and pesticide spraying periods affect the basic reproduction number.
10.3390/plants12203659
Function and molecular mechanism analysis of CaLasSDE460 effector involved in the pathogenesis of "Candidatus Liberibacter asiaticus" in citrus.
Molecular horticulture
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.
10.1186/s43897-023-00062-3
Sec-Delivered Effector 1 (SDE1) of ' Liberibacter asiaticus' Promotes Citrus Huanglongbing.
Clark Kelley J,Pang Zhiqian,Trinh Jessica,Wang Nian,Ma Wenbo
Molecular plant-microbe interactions : MPMI
Sec-delivered effector 1 (SDE1) from the huanglongbing (HLB)-associated bacterium ' Liberibacter asiaticus' was previously characterized as an inhibitor of defense-related, papain-like cysteine proteases in vitro and in planta. Here, we investigated the contributions of SDE1 to HLB progression. We found that SDE1 expression in the model plant caused severe yellowing in mature leaves, reminiscent of both ' L. asiaticus' infection symptoms and accelerated leaf senescence. Induction of senescence signatures was also observed in the SDE1-expressing lines. These signatures were apparent in older leaves but not in seedlings, suggesting an age-associated effect. Furthermore, independent lines of transgenic (L.) Macfadyen (Duncan grapefruit) that express exhibited hypersusceptibility to ' L. asiaticus'. Similar to , transgenic citrus expressing SDE1 showed altered expression of senescence-associated genes, but only after infection with ' L. asiaticus'. These findings suggest that SDE1 is a virulence factor that contributes to HLB progression, likely by inducing premature or accelerated senescence in citrus. This work provides new insight into HLB pathogenesis.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
10.1094/MPMI-05-20-0123-R
Potential of resistance inducers for citrus huanglongbing management via soil application and assessment of induction of pathogenesis-related protein genes.
Heliyon
Huanglongbing (HLB) or citrus greening currently is the most devastating citrus disease worldwide. Unfortunately, no practical cure has been available up to now. This makes the control of HLB as early as possible very important to be conducted. The objective of this study was to investigate the efficacy of the application of salicylic acid (SA) and Phenylacetic acid (PAA) on one-year-old seedlings of different citrus species (, , ) growing on and by soil drench methods. Factorial analysis of variance showed the percent change in " Liberibacter asiaticus" titer and disease severity on a different combination of citrus species growing on the two rootstocks treated with inducers and Oxytetracycline (OTC) were significantly different compared to the untreated plants. SA alone or in combination with OTC provided excellent (P-value < 0.05) control of HLB based on all parameters. The interaction between both factors (Rootstocks x Citrus species) significantly influenced the Ct value (P-value = 0.0001). " Liberibacter asiaticus" titer in plants treated with OTC was reduced significantly with a range of -18.75 up to -78.42. Overall, the highest reduction was observed in the application of OTC on sweet orange growing on (-78.42), while the lowest reduction was observed in the same cultivar which was treated with a combination of SA and OTC (-3.36). Induction of pathogenesis-related () genes, i.e., , , and , biosynthesis of Jasmonic acid and ethylene which are also important pathways to defense activity were also significantly increased in treated plants compared to untreated plants. This study suggests that the application of inducer alone is acceptable for HLB management. We proposed the application of SA and PAA as a soil drench on the citrus seedlings as promising, easy, and environmentally safe for HLB disease control on citrus seedlings.
10.1016/j.heliyon.2023.e19715
An effector from the Huanglongbing-associated pathogen targets citrus proteases.
Clark Kelley,Franco Jessica Yvette,Schwizer Simon,Pang Zhiqian,Hawara Eva,Liebrand Thomas W H,Pagliaccia Deborah,Zeng Liping,Gurung Fatta B,Wang Pengcheng,Shi Jinxia,Wang Yinsheng,Ancona Veronica,van der Hoorn Renier A L,Wang Nian,Coaker Gitta,Ma Wenbo
Nature communications
The citrus industry is facing an unprecedented challenge from Huanglongbing (HLB). All cultivars can be affected by the HLB-associated bacterium 'Candidatus Liberibacter asiaticus' (CLas) and there is no known resistance. Insight into HLB pathogenesis is urgently needed in order to develop effective management strategies. Here, we use Sec-delivered effector 1 (SDE1), which is conserved in all CLas isolates, as a molecular probe to understand CLas virulence. We show that SDE1 directly interacts with citrus papain-like cysteine proteases (PLCPs) and inhibits protease activity. PLCPs are defense-inducible and exhibit increased protein accumulation in CLas-infected trees, suggesting a role in citrus defense responses. We analyzed PLCP activity in field samples, revealing specific members that increase in abundance but remain unchanged in activity during infection. SDE1-expressing transgenic citrus also exhibit reduced PLCP activity. These data demonstrate that SDE1 inhibits citrus PLCPs, which are immune-related proteases that enhance defense responses in plants.
10.1038/s41467-018-04140-9
Comparative Proteomic Analysis of Sweet Orange Petiole Provides Insights Into the Development of Huanglongbing Symptoms.
Li Bo,Zhang Yi,Qiu Dewen,Francis Frédéric,Wang Shuangchao
Frontiers in plant science
Huanglongbing (HLB) is the most destructive citrus disease worldwide. This is associated with the phloem-limited bacterium Liberibacter, and the typical symptom is leaf blotchy mottle. To better understand the biological processes involved in the establishment of HLB disease symptoms, the comparative proteomic analysis was performed to reveal the global protein accumulation profiles in leaf petiole, where there are massive HLB pathogens of L. asiaticus-infected Newhall sweet orange () plants at the asymptomatic and symptomatic stages compared to their healthy counterpart. Photosynthesis, especially the pathway involved in the photosystem I and II light reactions, was shown to be suppressed throughout the whole L. asiaticus infection cycle. Also, starch biosynthesis was induced after the symptom-free prodromal period. Many defense-associated proteins were more extensively regulated in the petiole with the symptoms than the ones from healthy plants. The change of salicylic and jasmonic acid levels in different disease stages had a positive correlation with the abundance of phytohormone biosynthesis-related proteins. Moreover, the protein-protein interaction network analysis indicated that an F-type ATPase and an alpha-1,4 glucan phosphorylase were the core nodes in the interactions of differentially accumulated proteins. Our study indicated that the infected citrus plants probably activated the non-unified and lagging enhancement of defense responses against L. asiaticus at the expense of photosynthesis and contribute to find out the key L. asiaticus-responsive genes for tolerance and resistance breeding.
10.3389/fpls.2021.656997
' Liberibacter asiaticus' secretory protein SDE3 inhibits host autophagy to promote Huanglongbing disease in citrus.
Autophagy
Antimicrobial acroautophagy/autophagy plays a vital role in degrading intracellular pathogens or microbial molecules in host-microbe interactions. However, microbes evolved various mechanisms to hijack or modulate autophagy to escape elimination. Vector-transmitted phloem-limited bacteria, Liberibacter (. Liberibacter) species, cause Huanglongbing (HLB), one of the most catastrophic citrus diseases worldwide, yet contributions of autophagy to HLB disease proliferation remain poorly defined. Here, we report the identification of a virulence effector in ". Liberibacter asiaticus" (Las), SDE3, which is highly conserved among the ". Liberibacter". expression not only promotes the disease development of HLB and canker in sweet orange () plants but also facilitates and viral infections in , and ( ). SDE3 directly associates with citrus cytosolic glyceraldehyde-3-phosphate dehydrogenases (CsGAPCs), which negatively regulates plant immunity. Overexpression of and significantly inhibits autophagy in citrus, , and . Intriguingly, SDE3 undermines autophagy-mediated immunity by the specific degradation of CsATG8 family proteins in a CsGAPC1-dependent manner. CsATG8 degradation is largely rescued by treatment with an inhibitor of the late autophagic pathway, E64d. Furthermore, ectopic expression of enhances resistance. Collectively, these results suggest that SDE3-CsGAPC interactions modulate -mediated autophagy to enhance Las progression in citrus. ACP: asian citrus psyllid; ACD2: ACCELERATED CELL DEATH 2; ATG: autophagy related; . Liberibacter: Liberibacter; CaMV: cauliflower mosaic virus; CMV: cucumber mosaic virus; Cs: ; EV: empty vector; GAPC: cytosolic glyceraldehyde-3-phosphate dehydrogenase; HLB: huanglongbing; HO: hydrogen peroxide; Las: liberibacter asiaticus; Laf: liberibacter africanus; Lam: liberibacter americanus; : pv. tomato; PVX: potato virus X; ROS: reactive oxygen species; SDE3: sec-delivered effector 3; TEM: transmission electron microscopy; VIVE : virus-induced virulence effector; WT: wild-type; : subsp. .
10.1080/15548627.2023.2213040
A Pathogen Secreted Protein as a Detection Marker for Citrus Huanglongbing.
Frontiers in microbiology
The citrus industry is facing an unprecedented crisis due to Huanglongbing (HLB, aka citrus greening disease), a bacterial disease associated with the pathogen Liberibacter asiaticus (Las) that affects all commercial varieties. Transmitted by the Asian citrus psyllid (ACP), Las colonizes citrus phloem, leading to reduced yield and fruit quality, and eventually tree decline and death. Since adequate curative measures are not available, a key step in HLB management is to restrict the spread of the disease by identifying infected trees and removing them in a timely manner. However, uneven distribution of Las cells in infected trees and the long latency for disease symptom development makes sampling of trees for Las detection challenging. Here, we report that a Las secreted protein can be used as a biomarker for detecting HLB infected citrus. Proteins secreted from Las cells can presumably move along the phloem, beyond the site of ACP inoculation and Las colonized plant cells, thereby increasing the chance of detecting infected trees. We generated a polyclonal antibody that effectively binds to the secreted protein and developed serological assays that can successfully detect Las infection. This work demonstrates that antibody-based diagnosis using a Las secreted protein as the detection marker for infected trees offers a high-throughput and economic approach that complements the approved quantitative polymerase chain reaction-based methods to enhance HLB management programs.
10.3389/fmicb.2017.02041
Overexpression of the salicylic acid binding protein 2 (SABP2) from tobacco enhances tolerance against Huanglongbing in transgenic citrus.
Plant cell reports
KEY MESSAGE:Overexpression of the salicylic acid binding protein 2 (SABP2) gene from Tobacco results in enhanced tolerance to Huanglongbing (HLB; citrus greening disease) in transgenic sweet oranges. Huanglongbing (HLB), the most destructive citrus disease, is caused by Candidatus Liberibacter asiaticus (CaLas). Currently, no cure for this disease exists, and all commercially planted cultivars are highly susceptible. Salicylic Acid Binding Protein 2 (SABP2) is a well-characterized protein essential for establishing systemic acquired resistance (SAR) in tobacco. The constitutive over expression of SABP2 from tobacco (NtSABP2) in 'Hamlin' sweet orange resulted in the production of several transgenic lines with variable transcript levels. Transient expression of the NtSABP2-EGFP fusion protein in Nicotiana benthamiana plants demonstrated that NtSABP2 was cytosolic in its subcellular localization. In a long-term field study, we identified a SABP2 transgenic line with significantly reduced HLB symptoms that maintained a consistently low CaLas titer. Transcriptome analysis of this selected transgenic line demonstrated upregulation of several genes related to plant defense and SAR pathways. Genes, such as NPR family genes and those coding for monooxygenases and lipoxygenases, were upregulated in the 35S-NtSABP2 overexpressing line and might be candidates for incorporation into our citrus improvement program.
10.1007/s00299-022-02922-6
Engineering of citrus to obtain huanglongbing resistance.
Alquézar Berta,Carmona Lourdes,Bennici Stefania,Peña Leandro
Current opinion in biotechnology
Huanglongbing (HLB) disease is threatening the sustainability of citriculture in affected regions because of its rapid spread and the severity of the symptoms it induces. Herein, we summarise the main research findings that can be exploited to develop HLB-resistant cultivars. A major bottleneck has been the lack of a system for the ex vivo cultivation of HLB-associated bacteria (CLs) in true plant hosts, which precludes the evaluation of target genes/metabolites in reliable plant/pathogen/vector environments. With regard to HLB vectors, several biotechnologies which have been proven in laboratory settings to be effective for insect control are presented. Finally, new genotypes that are resistant to CLs or their insect vectors are described, and the most relevant strategies for fighting HLB are highlighted.
10.1016/j.copbio.2021.06.003
Controlling Citrus Huanglongbing: Green Sustainable Development Route Is the Future.
Frontiers in plant science
Huanglongbing (HLB) is the most severe bacterial disease of citrus crops caused by Liberibacter spp. It causes a reduction in fruit yield, poor fruit quality, and even plants death. Due to the lack of effective medicine, HLB is also called citrus "AIDS." Currently, it is essential for the prevention and control of HLB to use antibiotics and pesticides while reducing the spread of HLB by cultivating pathogen-free seedlings, removing disease trees, and killing Asian citrus psyllid (ACP). New compounds [e.g., antimicrobial peptides (AMPs) and nanoemulsions] with higher effectiveness and less toxicity were also found and they have made significant achievements. However, further evaluation is required before these new antimicrobial agents can be used commercially. In this review, we mainly introduced the current strategies from the aspects of physical, chemical, and biological and discussed their environmental impacts. We also proposed a green and ecological strategy for controlling HLB basing on the existing methods and previous research results.
10.3389/fpls.2021.760481
Dual Functions of a Stable Peptide against Citrus Huanglongbing Disease.
Wang Xu,Chen Jian,Liu Na,Fu Zheng Qing
Trends in plant science
The most prominent problem in the current citrus industry worldwide is the epidemic of citrus Huanglongbing (HLB), also known as greening disease. Huang et al. identified a stable peptide, which has antimicrobial activities and induces systemic immune response against HLB, from Australian finger lime. This peptide effectively suppresses disease symptoms in citrus and protects healthy trees against this disease.
10.1016/j.tplants.2021.04.006
A Calcium-Dependent Protein Kinase Regulates the Defense Response in .
Molecular plant-microbe interactions : MPMI
Citrus Huanglongbing (HLB), which is caused by ' Liberibacter asiaticus' (CLas), is one of the most destructive citrus diseases worldwide, and defense-related gene resources remain largely unexplored. Calcium signaling plays an important role in diverse biological processes. In plants, a few calcium-dependent protein kinases (CDPKs/CPKs) have been shown to contribute to defense against pathogenic microbes. The genome of encodes dozens of CPKs. In this study, the role of calcium-dependent protein kinases (CsCPKs) in defense was investigated. Silencing of compromised the induction of defense-related genes in . Expression of a constitutively active form of CsCPK6 (CsCPK6) triggered the activation of defense-related genes in . Complementation of CsCPK6 rescued the defense-related gene induction in an mutant, indicating that CsCPK6 carries CPK activity and is capable of functioning as a CPK in . Moreover, an effector derived from CLas inhibits defense induced by the expression of and autophosphorylation of CsCPK6, which suggests the involvement of CsCPK6 and calcium signaling in defense. These results support a positive role for CsCPK6 in defense against CLas, and the autoinhibitory regulation of CsCPK6 provides a potential genome-editing target for improving defense. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
10.1094/MPMI-12-23-0208-R
Metagenomic Analysis of Rhizospheric Bacterial Community of Citrus Trees Expressing Phloem-Directed Antimicrobials.
Microbial ecology
Huanglongbing, also known as citrus greening, is currently the most devastating citrus disease with limited success in prevention and mitigation. A promising strategy for Huanglongbing control is the use of antimicrobials fused to a carrier protein (phloem protein of 16 kDa or PP16) that targets vascular tissues. This study investigated the effects of genetically modified citrus trees expressing Citrus sinensis PP16 (CsPP16) fused to human lysozyme and β-defensin-2 on the soil microbiome diversity using 16S amplicon analysis. The results indicated that there were no significant alterations in alpha diversity, beta diversity, phylogenetic diversity, differential abundance, or functional prediction between the antimicrobial phloem-overexpressing plants and the control group, suggesting minimal impact on microbial community structure. However, microbiota diversity analysis revealed distinct bacterial assemblages between the rhizosphere soil and root environments. This study helps to understand the ecological implications of crops expressing phloem-targeted antimicrobials for vascular disease management, with minimal impact on soil microbiota.
10.1007/s00248-024-02408-w
" Liberibacter asiaticus" Secretes Nonclassically Secreted Proteins That Suppress Host Hypersensitive Cell Death and Induce Expression of Plant Pathogenesis-Related Proteins.
Applied and environmental microbiology
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. " 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 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 HO overaccumulation in , 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 genes, in particular and , could negatively regulate HR-based cell death development and HO 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 genes. 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.
10.1128/AEM.00019-21
Comparative Transcriptome and sRNAome Analysis Suggest Coordinated Citrus Immune Responses against Huanglongbing Disease.
Plants (Basel, Switzerland)
Citrus Huanglongbing (HLB), caused by the phloem-inhibiting bacterium Liberibacter asiaticus (Las), is the most devastating citrus disease, intimidating citrus production worldwide. Although commercially cultivated citrus cultivars are vulnerable to Las infection, HLB-tolerant attributes have, however, been observed in certain citrus varieties, suggesting a possible pathway for identifying innate defense regulators that mitigate HLB. By adopting transcriptome and small RNAome analysis, the current study compares the responses of HLB-tolerant lemon ( L.) with HLB-susceptible Shatangju mandarin ( Blanco cv. Shatangju) against Las infection. Transcriptome analysis revealed significant differences in gene expression between lemon and Shatangju. A total of 1751 and 3076 significantly differentially expressed genes were identified in Shatangju and lemon, respectively. Specifically, Las infected lemon tissues demonstrated higher expressions of genes involved in antioxidant enzyme activity, protein phosphorylation, carbohydrate, cell wall, and lipid metabolism than Shatangju. Wet-lab experiments further validated these findings, demonstrating increased antioxidant enzyme activity in lemon: APX (35%), SOD (30%), and CAT (64%) than Shatangju. Conversely, Shatangju plants exhibited higher levels of oxidative stress markers like HO (44.5%) and MDA content (65.2%), alongside pronounced ion leakage (11.85%), than lemon. Moreover, microscopic investigations revealed that Las infected Shatangju phloem exhibits significantly more starch and callose accumulation than lemon. Furthermore, comparative sRNA profiles revealed the potential defensive regulators for HLB tolerance. In Shatangju, increased expression of csi-miR166 suppresses the expression of disease-resistant proteins, leading to inadequate defense against Las. Conversely, reduced expression of csi-miR166 in lemon plants enables them to combat HLB by activating disease-resistance proteins. The above findings indicate that when infected with Las, lemon exhibits stronger antioxidative activity and higher expression of disease-resistant genes, contributing to its enhanced tolerance to HLB. In contrast, Shatangju shows lower antioxidative activity, reduced expression of disease-resistant genes, significant ion leakage, and extensive callose deposition, possibly related to damage to plant cell structure and blockage of phloem sieve tubes, thereby promoting the development of HLB symptoms.
10.3390/plants13111496
SDE19, a SEC-dependent effector from 'Candidatus Liberibacter asiaticus' suppresses plant immunity and targets Citrus sinensis Sec12 to interfere with vesicle trafficking.
PLoS pathogens
Citrus huanglongbing (HLB), which is caused by the phloem-colonizing bacteria Candidatus Liberibacter asiaticus (CLas), poses a significant threat to citrus production worldwide. The pathogenicity mechanism of HLB remains poorly understood. SEC-dependent effectors (SDEs) have been suggested to play critical roles in the interaction between citrus and CLas. Here, we explored the function of CLIBASIA_05320 (SDE19), a core SDE from CLas, and its interaction with its host target. Our data revealed that SDE19 is expressed at higher level during infection of citrus than that during infection of the Asian citrus psyllid. Subcellular localization assays showed that SDE19 is localized in the nucleus and cytoplasm and is capable of moving from cell to cell in Nicotiana benthamiana. To investigate whether SDE19 facilitates pathogen infection, we generated transgenic Arabidopsis thaliana and citrus plants overexpressing SDE19. Transgenic A. thaliana and citrus plants were more susceptible to Pseudomonas syringae pv. tomato (Pst) and Xanthomonas citri subsp. citri (Xcc), respectively. In addition, RNA-seq analysis demonstrated that overexpression of SDE19 resulted in a reprogramming of expression of genes related to biotic stimulus responses. SDE19 interacts with Citrus sinensis Sec12, a guanine nucleotide exchange factor responsible for the assembly of plant COPII (coat protein II)-coated vesicles, which mediate vesicle trafficking from the ER to the Golgi. SDE19 colocalizes with Sec12 in the ER by binding to its N-terminal catalytic region, affecting the stability of Sec12 through the 26S proteasome. This interaction hinders the secretion of apoplastic defense-related proteins such as PR1, P69B, GmGIP1, and RCR3. Furthermore, the secretion of PR1 and callose deposition is decreased in SDE19-transgenic A. thaliana. Taken together, SDE19 is a novel virulent SDE secreted by CLas that interacts with Sec12 to disrupt vesicle trafficking, inhibit defense-related proteins secretion, and promote bacterial infection. This study sheds light on how CLas manipulates the host vesicle trafficking pathway to suppress the secretion of defense-related proteins and interfere with plant immunity.
10.1371/journal.ppat.1012542
A prophage-encoded nonclassical secretory protein of "Candidatus Liberibacter asiaticus" induces a strong immune response in Nicotiana benthamiana and citrus.
Molecular plant pathology
Huanglongbing (HLB), associated with "Candidatus Liberibacter asiaticus" (CLas), is a globally devastating plant disease. The highly reduced genome of CLas encodes a number of secretory proteins. The conserved prophage-encoded protein AGH17470 is herein identified as a nonclassical secretory protein. We confirmed that the N-terminal and C-terminal sequences jointly determine the secretion of AGH17470. The transient expression of AGH17470 protein in Nicotiana benthamiana caused hypersensitive response (HR) cell death in infiltrated leaves and systemically infected leaves as well as the dwarfing of the entire plant, suggesting that AGH17470 is involved in the plant immune response, growth, and development. Overexpression of AGH17470 in N. benthamiana and citrus plants up-regulated the transcription of pathogenesis-related and salicylic acid (SA)-signalling pathway genes and promoted SA accumulation. Furthermore, transient expression of AGH17470 enhanced the resistance of sweet orange to Xanthomonas citri subsp. citri. To our knowledge, AGH17470 is the first prophage-encoded secretory protein demonstrated to elicit an HR and induce a strong plant immune response. The findings have increased our understanding of prophage-encoded secretory protein genes, and the results provide clues as to the plant defence response against CLas.
10.1111/mpp.13206
Transcriptome sequencing and ITRAQ reveal the detoxification mechanism of Bacillus GJ1, a potential biocontrol agent for Huanglongbing.
Tang Jizhou,Ding Yuanxi,Nan Jing,Yang Xiangyu,Sun Liang,Zhao Xiuyun,Jiang Ling
PloS one
Huanglongbing (HLB) is the most serious disease affecting citrus production worldwide. No HLB-resistant citrus varieties exist. The HLB pathogen Candidatus Liberibacter asiaticus is nonculturable, increasing the difficulty of preventing and curing the disease. We successfully screened the biocontrol agent Bacillus GJ1 for the control of HLB in nursery-grown citrus plants. RNA sequencing (RNA-seq) of the transcriptome and isobaric tags for relative and absolute quantification of the proteome revealed differences in the detoxification responses of Bacillus GJ1-treated and -untreated Ca. L. asiaticus-infected citrus. Phylogenetic tree alignment showed that GJ1 was classified as B. amyloliquefaciens. The effect of eliminating the HLB pathogen was measured using real-time quantitative polymerase chain reaction (qPCR) and PCR. The results indicate that the rate of detoxification reached 50% after seven irrigations, of plants with an OD600nm≈1 Bacillus GJ1 suspension. Most importantly, photosynthesis-antenna proteins, photosynthesis, plant-pathogen interactions, and protein processing in the endoplasmic reticulum were significantly upregulated (padj < 0.05), as shown by the KEGG enrichment analysis of the transcriptomes; nine of the upregulated genes were validated by qPCR. Transcription factor analysis of the transcriptomes was performed, and 10 TFs were validated by qPCR. Cyanoamino acid metabolism, regulation of autophagy, isoflavonoid biosynthesis, starch and sucrose metabolism, protein export, porphyrin and chlorophyll metabolism, and carotenoid biosynthesis were investigated by KEGG enrichment analysis of the proteome, and significant differences were found in the expression of the genes involved in those pathways. Correlation analysis of the proteome and transcriptome showed common entries for the significantly different expression of proteins and the significantly different expression of genes in the GO and KEGG pathways, respectively. The above results reveal important information about the detoxification pathways.
10.1371/journal.pone.0200427
Peptidomics Approaches for the Identification of Bioactive Molecules from .
Fleites Laura A,Johnson Richard,Kruse Angela R,Nachman Ronald J,Hall David G,MacCoss Michael,Heck Michelle L
Journal of proteome research
Huanglongbing (HLB), a deadly citrus disease, is primarily associated with Liberibacter asiaticus (CLas) and spread by the hemipteran insect . Control strategies to combat HLB are urgently needed. In this work, we developed and compared workflows for the extraction of the peptidome, a dynamic set of polypeptides produced by proteolysis and other cellular processes. High-resolution mass spectrometry revealed bias among methods reflecting the physiochemical properties of the peptides: while TCA/acetone-based methods resulted in enrichment of C-terminally amidated peptides, a modification characteristic of bioactive peptides, larger peptides were overrepresented in the aqueous phase of chloroform/methanol extracts, possibly indicative of reduced co-analytical degradation during sample preparation. Parallel reaction monitoring (PRM) was used to validate the structure and upregulation of peptides derived from hemocyanin, a immune system protein, in insects reared on healthy and CLas-infected trees. Mining of the data sets also revealed 122 candidate neuropeptides, including PK/PBAN family neuropeptides and kinins, biostable analogs of which have known insecticidal properties. Taken together, this information yields new, in-depth insights into peptidomics methodology. Additionally, the putative neuropeptides identified may lead to psyllid mortality if applied to or expressed in citrus, consequently blocking the spread of HLB disease in citrus groves.
10.1021/acs.jproteome.9b00509
Liberibacter: From Movement, Host Responses, to Symptom Development of Citrus Huanglongbing.
Pandey Sheo Shankar,Hendrich Connor,Andrade Maxuel O,Wang Nian
Phytopathology
Liberibacter spp. are fastidious α-proteobacteria that cause multiple diseases on plant hosts of economic importance, including the most devastating citrus disease: Huanglongbing (HLB). HLB was reported in Asia a century ago but has since spread worldwide. Understanding the pathogenesis of Liberibacter spp. remains challenging as they are yet to be cultured in artificial media and infect the phloem, a sophisticated environment that is difficult to manipulate. Despite those challenges, tremendous progress has been made on . Liberibacter pathosystems. Here, we first reviewed recent studies on genetic information of flagellar and type IV pili biosynthesis, their expression profiles, and movement of Liberibacter spp. inside the plant and insect hosts. Next, we reviewed the transcriptomic, proteomic, and metabolomic studies of susceptible and tolerant plant genotypes to . Liberibacter spp. infection and how . Liberibacter spp. adapt in plants. Analyses of the interactions between plants and . Liberibacter spp. imply the involvement of immune response in the . Liberibacter pathosystems. Lastly, we reviewed how . Liberibacter spp. movement inside and interactions with plants lead to symptom development.
10.1094/PHYTO-08-21-0354-FI
Molecular Mechanisms of Effector Proteins: A Comprehensive Review.
Infection and drug resistance
can be categorized into many serotypes, which are specific to known hosts or broadhosts. It makes no difference which one of the serotypes would penetrate the gastrointestinal tract because they all face similar obstacles such as mucus and microbiome. However, following their penetration, some species remain in the gastrointestinal tract; yet, others spread to another organ like gallbladder. is required to alter the immune response to sustain its intracellular life. Changing the host response requires particular effector proteins and vehicles to translocate them. To this end, a categorized gene called pathogenicity island (SPI) was developed; genes like pathogenicity island encode aggressive or modulating proteins. Initially, needs to be attached and stabilized via adhesin factor, without which no further steps can be taken. In this review, an attempt has been made to elaborate on each factor attached to the host cell or to modulating and aggressive proteins that evade immune systems. This review includes four sections: (A) attachment factors or T3SS- independent entrance, (B) effector proteins or T3SS-dependent entrance, (c) regulation of invasive genes, and (D) regulation of immune responses.
10.2147/IDR.S230604
Accurate prediction of huanglongbing occurrence in citrus plants by machine learning-based analysis of symbiotic bacteria.
Frontiers in plant science
Huanglongbing (HLB), the most prevalent citrus disease worldwide, is responsible for substantial yield and economic losses. Phytobiomes, which have critical effects on plant health, are associated with HLB outcomes. The development of a refined model for predicting HLB outbreaks based on phytobiome markers may facilitate early disease detection, thus enabling growers to minimize damages. Although some investigations have focused on differences in the phytobiomes of HLB-infected citrus plants and healthy ones, individual studies are inappropriate for generating common biomarkers useful for detecting HLB on a global scale. In this study, we therefore obtained bacterial information from several independent datasets representing hundreds of citrus samples from six continents and used these data to construct HLB prediction models based on 10 machine learning algorithms. We detected clear differences in the phyllosphere and rhizosphere microbiomes of HLB-infected and healthy citrus samples. Moreover, phytobiome alpha diversity indices were consistently higher for healthy samples. Furthermore, the contribution of stochastic processes to citrus rhizosphere and phyllosphere microbiome assemblies decreased in response to HLB. Comparison of all constructed models indicated that a random forest model based on 28 bacterial genera in the rhizosphere and a bagging model based on 17 bacterial species in the phyllosphere predicted the health status of citrus plants with almost 100% accuracy. Our results thus demonstrate that machine learning models and phytobiome biomarkers may be applied to evaluate the health status of citrus plants.
10.3389/fpls.2023.1129508
Early detection of Huanglongbing with EESI-MS indicates a role of phenylpropanoid pathway in citrus.
Xue Ahui,Liu Yongquan,Li Haoxing,Cui Meng,Huang Xueyong,Wang Wenjing,Wu Dong,Guo Xiali,Hao Yingbin,Luo Liping
Analytical biochemistry
Huanglongbing (HLB), a devastating disease for citrus worldwide, is caused by Candidatus Liberibacter asiaticus (CLas). In this study, we employed a novel extractive electrospray ionization-mass spectrometry (EESI-MS) method to analyze the metabolites in leaves of uninfected and HLB-infected Newhall navel orange. The results showed that uninfected and HLB-infected leaves could be readily distinguished based on EESI-MS combined by multivariable analysis. Nine phenolic compounds involved in phenylpropanoid pathway, such as p-coumaric acid, naringin, and apigenin, were principal components to distinguish the leaves of uninfected and HLB-infected Newhall navel orange. Gene expression was also conducted to further explore the molecular mechanism of phenylpropanoid branch pathway in HLB. The expression of genes (4CL, HCT, CHI, CHS, CYP, and C12R) involved in phenylpropanoid branch pathway was increased in asymptomatic and early period of HLB-infected leaves, while decreased in later period of HLB-infected leaves. This study provides a novel method for early detection of citrus HLB and suggests the regulation mechanism of phenylpropanoid pathway in the interaction between citrus and CLas.
10.1016/j.ab.2021.114511
LasΔ5315 Effector Induces Extreme Starch Accumulation and Chlorosis as . Liberibacter asiaticus Infection in .
Frontiers in plant science
Huanglongbing (HLB), a destructive plant bacterial disease, severely impedes worldwide citrus production. HLB is associated with a phloem-limited α-proteobacterium, Liberibacter asiaticus (Las). Las infection causes yellow shoots and blotchy mottle on leaves and is associated with excessive starch accumulation. However, the mechanisms underlying the starch accumulation remain unknown. We previously showed that the Las5315mp effector induced callose deposition and cell death in . In this study, we demonstrated that Las can experimentally infect via dodder transmission. Furthermore, we revealed another key function of the Las5315 effector by demonstrating that transient expression of the truncated form of the effector, LasΔ5315, induced excessive starch accumulation by 6 fold after 8 dpi in after removal of the chloroplast transit peptide from the Las5315mp. The induction mechanisms of LasΔ5315 in were attributed to the up-regulation of ADP-glucose pyrophosphorylase, granule-bound starch synthase, soluble starch synthase, and starch branching enzyme for increasing starch production, and to the significant down-regulation of the starch degradation enzymes: alpha-glucosidase, alpha-amylase, and glycosyl hydrolase for decreasing starch degradation. This is the first report that Las can infect the model plant . Using this model plant, we demonstrated that the LasΔ5315 effector caused the most prominent HLB symptoms, starch accumulation and chlorosis as Las infection in . Altogether the Las 5315 effector is critical for Las pathogenesis, and therefore, an important target for interference.
10.3389/fpls.2018.00113
Citrus CsACD2 Is a Target of Candidatus Liberibacter Asiaticus in Huanglongbing Disease.
Pang Zhiqian,Zhang Li,Coaker Gitta,Ma Wenbo,He Sheng-Yang,Wang Nian
Plant physiology
Citrus Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (Las), is one of the most destructive citrus diseases worldwide, yet how Las causes HLB is poorly understood. Here we show that a Las-secreted protein, SDE15 (CLIBASIA_04025), suppresses plant immunity and promotes Las multiplication. Transgenic expression of SDE15 in Duncan grapefruit (Citrus × paradisi) suppresses the hypersensitive response induced by Xanthomonas citri ssp. citri (Xcc) and reduces the expression of immunity-related genes. SDE15 also suppresses the hypersensitive response triggered by the Xanthomonas vesicatoria effector protein AvrBsT in Nicotiana benthamiana, suggesting that it may be a broad-spectrum suppressor of plant immunity. SDE15 interacts with the citrus protein CsACD2, a homolog of Arabidopsis (Arabidopsis thaliana) ACCELERATED CELL DEATH 2 (ACD2). SDE15 suppression of plant immunity is dependent on CsACD2, and overexpression of CsACD2 in citrus suppresses plant immunity and promotes Las multiplication, phenocopying overexpression of SDE15. Identification of CsACD2 as a susceptibility target has implications in genome editing for novel plant resistance against devastating HLB.
10.1104/pp.20.00348
Two Liberibacter Proteins Combine to Suppress Critical Innate Immune Defenses in Citrus.
Frontiers in plant science
We adopted a systems-based approach to determine the role of two Liberibacter asiaticus (Las) proteins, and Effector 3, in Huanglongbing (HLB) pathogenesis. While a published work suggests the involvement of these Las proteins HLB pathogenesis, the exact structure-based mechanism of their action has not been elucidated. We conducted the following experiments to determine the structure-based mechanisms of action. First, we immunoprecipitated the interacting citrus protein partners of and Effector 3 from the healthy and Las-infected Hamlin extracts and identified them by Liquid Chromatography with tandem mass spectrometry (LC-MS/MS). Second, we performed a split green fluorescent protein (GFP) assay in tobacco to validate that the interactions observed are also retained . The notable citrus targets of and Effector 3 include citrus innate immune proteins. Third, and studies were performed to show that and Effector 3 interact with and inhibit the functions of multiple citrus proteins belonging to the innate immune pathways. These inhibitory interactions led to a high level of reactive oxygen species, blocking of bactericidal lipid transfer protein (LTP), and induction of premature programed cell death (PCD), all of which are beneficial to Las lifecycle and HLB pathogenesis. Finally, we performed molecular dynamics simulations to visualize the interactions of and Effector 3, respectively, with LTP and Kunitz protease inhibitor. This led to the design of an LTP mimic, which sequestered and blocked and rescued the bactericidal activity of LTP thereby proving that , indeed, participates in HLB pathogenesis.
10.3389/fpls.2022.869178
A " Liberibacter asiaticus"-secreted polypeptide suppresses plant immune responses in and .
Frontiers in plant science
Citrus Huanglongbing (HLB), known as the most economically devastating disease in citrus industry, is mainly caused by phloem-restricted Gram-negative bacterium " Liberibacter asiaticus" (Las). To date, Las is still unculturable , which has been dramatically delaying the research on its pathogenesis, and only few Sec-dependent effectors (SDEs) have been identified to elucidate the pathogenesis of Las. Here, we confirmed that a Las-secreted Sec-dependent polypeptide, namely SECP8 (CLIBASIA_05330), localized in nucleus, cytoplasm and cytoplasmic membrane, and showed remarkably higher transcript abundance in citrus than in psyllids. (PVX)-mediated transient expression assays indicated that mSECP8 (the mature form of SECP8) suppressed pro-apoptotic mouse protein BAX and elicitin INF1-triggered hypersensitive response (HR) associated phenotypes, including cell death, HO accumulation and callose deposition. Intriguingly, mSECP8 also inhibited SDE1 (CLIBASIA_05315)-induced water-soaked and dwarfing symptoms in . In addition, mSECP8 can promote the susceptibility of transgenic Wanjincheng orange () to Las invasion and further HLB symptom development, and it contributes to the proliferation of subsp. (). Moreover, the expression of ten immunity-related genes were significantly down-regulated in mSECP8 transgenic citrus than those in wide-type (WT) plants. Overall, we propose that mSECP8 may serve as a novel broad-spectrum suppressor of plant immunity, and provide the first evidence counteractive effect among Las effectors. This study will enrich and provide new evidences for elucidating the pathogenic mechanisms of Las in citrus host.
10.3389/fpls.2022.997825