Transcriptional response of susceptible and tolerant citrus to infection with Candidatus Liberibacter asiaticus
ABSTRACT: Candidatus Liberibacter asiaticus (Las), a non-culturable phloem-limited bacterium, is the suspected causal agent of Huanglongbing (HLB) in Florida. HLB is one of the most devastating diseases of citrus and no resistant cultivars have been identified to date, though tolerance has been suggested in the genus Poncirus. A recent study conducted in our laboratory demonstrated tolerance of US-897, a hybrid of Poncirus trifoliata (L. Raf.) and the susceptible ‘Cleopatra’ mandarin (Citrus reticulata Blanco), to Ca. L. asiaticus, the presumed causal agent of HLB in Florida (Albrecht & Bowman, HortScience 46 (2011) 16-22). This study compares transcriptional changes in tolerant US-897 and susceptible ‘Cleopatra’ mandarin seedlings in response to infection with Las using the Affymetrix GeneChip citrus array with the main objective of identifying genes associated with tolerance to HLB. Such genes may be suitable as potential targets for biotechnology approaches, providing one strategy to possibly control this destructive disease of citrus. Fifteen months-old seedlings of the genotypes 'Cleopatra' mandarin and US-897 were graft-inoculated with non-infected (control) or Las-infected tissue from greenhouse-grown 'Valencia' (C. sinensis L.) plants. Six non-infected control plants each from 'Cleopatra' (MC) and US-897 (TC) and six infected plants each from Cleopatra (MI) and US-897 (TI) were used. Four to six leaves per plant were excised at 32 weeks after inoculation (wai) and immediately frozen in liquid nitrogen. Total RNA was extracted from all 24 plants. Equal amounts of RNA from two samples were pooled to obtain three biological replicates per genotype and treatment and used for hybridization on Affymetrix citrus microarrays.
Project description:Huanglongbing (HLB) (=citrus greening) is a destructive disease of citrus which is caused by a fastidious, phloem-inhabiting bacterium of the genus Candidatus Liberibacter. Large-scale analysis of gene expression changes in ‘Valencia’ orange leaves were studied during the course of 19 weeks after inoculation with Ca. L. asiaticus using the Affymetrix GeneChip® citrus genome array to provide new insights into the molecular basis of citrus response to this pathogen. Of the more than 33,000 probe sets on the microarray 21,067 were expressed in the leaves, of which 279 and 515 were differentially expressed (FDR ≤ 0.05) five to nine and 13-17 weeks after inoculation, respectively. Results from semi-quantitative RT-PCR analysis performed on 14 selected genes were highly correlated with those observed with the microarray. Gene expression changes involved a variety of different processes including cell defense, transport, cellular organization, photosynthesis, and carbohydrate metabolism. Notable was the pathogen-induced accumulation of transcripts for a phloem-specific lectin PP2-like protein. Transcriptional changes and their relation to disease symptom development are discussed. This is the first study of transcriptional profiling in citrus in response to liberibacter infection using microarray technology. Huanglongbing (HLB) is a destructive disease of citrus which is suspected to be caused by a phloem-inhabiting bacterium of the genus Candidatus Liberibacter. Large-scale analysis of gene expression changes in ‘Valencia’ orange (C. sinensis) leaves were studied during the course of 19 weeks after inoculation with Ca. L. asiaticus (Las), the pathogen associated with HLB in Florida, using the Affymetrix GeneChip® citrus genome array to provide new insights into the molecular basis of citrus response to this pathogen. Three year-old 'Valencia' orange (C. sinensis) scions on Cleopatra mandarin (C. reticulata Blanco) rootstocks were graft-inoculated with non-infected (control) or Las-infected tissue from greenhouse- (control) or field-grown 'Lisbon' lemon (C. limon) trees. Fully expanded leaves were collected at 5, 9, 13, and 17 weeks after inoculation (wai). Total RNA was extracted from two control plants and from three Las-inoculated plants per time point. Equal amounts of RNA were combined from samples collected 5 wai and 9 wai and from samples collected 13 wai and 17 wai, resulting in two non-infected and three infected biological replicates for both early (5-9 wai) and late (13-17 wai) time points and used for hybridization on Affymetrix citrus microarrays.
Project description:Citrus Huanglongbing (HLB) associated with 'Candidatus Liberibacter asiaticus' (Las) and citrus canker disease incited by Xanthomonas citri are the most devastating citrus diseases worldwide. To control citrus HLB and canker disease, we previously screened over forty antimicrobial peptides (AMPs) in vitro for their potential application in genetic engineering. D2A21 was one of the most active AMPs against X. citri, Agrobacterium tumefaciens and Sinorhizobium meliloti with low hemolysis activity. Therefore, we conducted this work to assess transgenic expression of D2A21 peptide to achieve citrus resistant to canker and HLB. We generated a construct expressing D2A21 and initially transformed tobacco as a model plant. Transgenic tobacco expressing D2A21 was obtained by Agrobacterium-mediated transformation. Successful transformation and D2A21 expression was confirmed by molecular analysis. We evaluated disease development incited by Pseudomonas syringae pv. tabaci in transgenic tobacco. Transgenic tobacco plants expressing D2A21 showed remarkable disease resistance compared to control plants. Therefore, we performed citrus transformations with the same construct and obtained transgenic Carrizo citrange. Gene integration and gene expression in transgenic plants were determined by PCR and RT-qPCR. Transgenic Carrizo expressing D2A21 showed significant canker resistance while the control plants showed clear canker symptoms following both leaf infiltration and spray inoculation with X. citri 3213. Transgenic Carrizo plants were challenged for HLB evaluation by grafting with Las infected rough lemon buds. Las titer was determined by qPCR in the leaves and roots of transgenic and control plants. However, our results showed that transgenic plants expressing D2A21 did not significantly reduce Las titer compared to control plants. We demonstrated that transgenic expression of D2A21 conferred resistance to diseases incited by P. syringae pv. tabaci and X. citri but not Las. Our results underscore the difficulty in controlling HLB compared to other bacterial diseases.
Project description:Huanglongbing (HLB) is a devastating citrus disease that is associated with bacteria of the genus 'Candidatus Liberibacter' (Ca. L.). Powerful diagnostic tools and management strategies are desired to control HLB. Host small RNAs (sRNA) play a vital role in regulating host responses to pathogen infection and are used as early diagnostic markers for many human diseases, including cancers. To determine whether citrus sRNAs regulate host responses to HLB, sRNAs were profiled from Citrus sinensis 10 and 14 weeks post grafting with Ca. L. asiaticus (Las)-positive or healthy tissue. Ten new microRNAs (miRNAs), 76 conserved miRNAs, and many small interfering RNAs (siRNAs) were discovered. Several miRNAs and siRNAs were highly induced by Las infection, and can be potentially developed into early diagnosis markers of HLB. miR399, which is induced by phosphorus starvation in other plant species, was induced specifically by infection of Las but not Spiroplasma citri that causes citrus stubborn-a disease with symptoms similar to HLB. We found a 35% reduction of phosphorus in Las-positive citrus trees compared to healthy trees. Applying phosphorus oxyanion solutions to HLB-positive sweet orange trees reduced HLB symptom severity and significantly improved fruit production during a 3-year field trial in south-west Florida. Our molecular, physiological, and field data suggest that phosphorus deficiency is linked to HLB disease symptomology.
Project description:Huanglongbing (HLB) or citrus greening is highly destructive disease that is affecting the citrus industry worldwide and it has killed millions of citrus plants globally. HLB is caused by the phloem limited, Gram negative, non-culturable, alpha-proteobacterium, 'Candidatus Liberibacter asiaticus'. Currently, polymerase chain reaction (PCR) and real time PCR have been the gold standard techniques used for detection of 'Ca. L. asiaticus'. These diagnostic methods are expensive, require well equipped laboratories, not user-friendly and not suitable for on-site detection of the pathogen. In this study, a sensitive, reliable, quick and low cost recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) technique has been developed as a diagnostic tool for detection of 'Ca. L. asiaticus'. The assay was standardized by designing the specific primer pair and probe based on the conserved 16S rRNA gene of 'Ca. L. asiaticus'. The assay was optimized for temperature and reaction time by using purified DNA and crude plant extracts and the best HLB-RPA-LFA was achieved at the isothermal temperature of 38°C for 20 to 30 min. The efficacy and sensitivity of the assay was carried out by using field grown, HLB-infected, HLB-doubtful and healthy citrus cultivars including mandarin, sweet orange cv. mosambi, and acid lime. The HLB-RPA-LFA did not show cross-reactivity with other citrus pathogens and is simple, cost-effective, rapid, user-friendly and sensitive. Thus, the HLB-RPA-LFA method has great potential to provide an improved diagnostic tool for detection of 'Ca. L. asiaticus' for the farmers, nurserymen, disease surveyors, mobile plant pathology laboratories, bud-wood certification and quarantine programs.
Project description:Huanglongbing (HLB), also known as citrus greening, is one of the most destructive diseases of citrus worldwide. HLB is associated with three species of 'Candidatus Liberibacter' with 'Ca. L. asiaticus' (Las) being the most widely distributed around the world, and the only species detected in Thailand. To understand the genetic diversity of Las bacteria in Thailand, we evaluated two closely-related effector genes, lasAI and lasAII, found within the Las prophages from 239 infected citrus and 55 infected psyllid samples collected from different provinces in Thailand. The results indicated that most of the Las-infected samples collected from Thailand contained at least one prophage sequence with 48.29% containing prophage 1 (FP1), 63.26% containing prophage 2 (FP2), and 19.38% containing both prophages. Interestingly, FP2 was found to be the predominant population in Las-infected citrus samples while Las-infected psyllids contained primarily FP1. The multiple banding patterns that resulted from amplification of lasAI imply extensive variation exists within the full and partial repeat sequence while the single band from lasAII indicates a low amount of variation within the repeat sequence. Phylogenetic analysis of Las-infected samples from 22 provinces in Thailand suggested that the bacterial pathogen may have been introduced to Thailand from China and the Philippines. This is the first report evaluating the genetic variation of a large population of Ca. L. asiaticus infected samples in Thailand using the two effector genes from Las prophage regions.
Project description:Huanglongbing (HLB or citrus greening disease) caused by Candidatus Liberibacter asiaticus (Las) is a great threat to the US citrus industry. There are no proven strategies to eliminate HLB disease and no cultivar has been identified with strong HLB resistance. Citrus canker is also an economically important disease associated with a bacterial pathogen (Xanthomonas citri). In this study, we characterized endogenous citrus thionins and investigated their expression in different citrus tissues. Since no HLB-resistant citrus cultivars have been identified, we attempted to develop citrus resistant to both HLB and citrus canker through overexpression of a modified plant thionin. To improve effectiveness for disease resistance, we modified and synthesized the sequence encoding a plant thionin and cloned into the binary vector pBinPlus/ARS. The construct was then introduced into Agrobacterium strain EHA105 for citrus transformation. Transgenic Carrizo plants expressing the modified plant thionin were generated by Agrobacterium-mediated transformation. Successful transformation and transgene gene expression was confirmed by molecular analysis. Transgenic Carrizo plants expressing the modified thionin gene were challenged with X. citri 3213 at a range of concentrations, and a significant reduction in canker symptoms and a decrease in bacterial growth were demonstrated compared to nontransgenic plants. Furthermore, the transgenic citrus plants were challenged with HLB via graft inoculation. Our results showed significant Las titer reduction in roots of transgenic Carrizo compared with control plants and reduced scion Las titer 12 months after graft inoculation. These data provide promise for engineering citrus disease resistance against HLB and canker.
Project description:BACKGROUND: Huanglongbing (HLB) is a highly destructive citrus disease which threatens citrus production worldwide and 'Candidatus Liberibacter asiaticus' (Las), a non-culturable phloem-limited bacterium, is an associated causal agent of the disease. To better understand the physiological and molecular processes involved in host responses to Las, 2-DE and mass spectrometry analyses, as well as ICP spectroscopy analysis were employed to elucidate the global protein expression profiles and nutrient concentrations in leaves of Las-infected grapefruit plants at pre-symptomatic or symptomatic stages for HLB. RESULTS: This study identified 123 protein spots out of 191 spots that showed significant changes in the leaves of grapefruit plants in response to Las infection and all identified spots matched to 69 unique proteins/peptides. A down-regulation of 56 proteins including those associated with photosynthesis, protein synthesis, and metabolism was correlated with significant reductions in the concentrations of Ca, Mg, Fe, Zn, Mn, and Cu in leaves of grapefruit plants in response to Las infection, particularly in symptomatic plants. Oxygen-evolving enhancer (OEE) proteins, a PSI 9 kDa protein, and a Btf3-like protein were among a small group of proteins that were down-regulated in both pre-symptomatic and symptomatic plants in response to Las infection. Furthermore, a Las-mediated up-regulation of 13 grapefruit proteins was detected, which included Cu/Zn superoxide dismutase, chitinases, lectin-related proteins, miraculin-like proteins, peroxiredoxins and a CAP 160 protein. Interestingly, a Las-mediated up-regulation of granule-bound starch synthase was correlated with an increase in the K concentrations of pre-symptomatic and symptomatic plants. CONCLUSIONS: This study constitutes the first attempt to characterize the interrelationships between protein expression and nutritional status of Las-infected pre-symptomatic or symptomatic grapefruit plants and sheds light on the physiological and molecular mechanisms associated with HLB disease development.
Project description:Huanglongbing (HLB), the most destructive citrus disease, is caused by three species of phloem-limited Candidatus Liberibacter. Chemical control is a critical short-term strategy against Candidatus Liberibacter asiaticus (Las). Currently, application of antibiotics in agricultural practices is limited due to public concerns regarding emergence of antibiotic-resistant bacteria and potential side effects in humans. The present study screened 39 antimicrobials (non-antibiotics) for effectiveness against Las using an optimized graft-based screening system. Results of principal component, hierarchical clustering and membership function analyses demonstrated that 39 antimicrobials were clustered into three groups: "effective" (Group I), "partly effective" (Group II), and "ineffective" (Group III). Despite different modes of action, 8 antimicrobials (aluminum hydroxide, D,L-buthionine sulfoximine, nicotine, surfactin from Bacillus subtilis, SilverDYNE, colloidal silver, EBI-601, and EBI-602), were all as highly effective at eliminating or suppressing Las, showing both the lowest Las infection rates and titers in treated scions and inoculated rootstock. The ineffective group, which included 21 antimicrobials, did not eliminate or suppress Las, resulting in plants with increased titers of Candidatus Liberibacter. The other 10 antimicrobials partly eliminated/suppressed Las in treated and graft-inoculated plants. These effective antimicrobials are potential candidates for HLB control either via rescuing infected citrus germplasms or restricted field application.
Project description:HLB is suggested to be caused by the phloem-limited fastidious prokaryotic α-proteobacterium “Candidatus Liberibacter spp.” Previous studies focused on the proteome and transcriptome analyses of citrus 5 to 35-week-after “Ca. L. spp.” inoculation. In this study, gene expression profiles was analyzed using mandarin of Citrus reticulate Blanco cv. jiaogan leaves after 2-year infection with “Ca. L. asiaticus”. The Affymetrix GeneChip® citrus genome were applied to study the molecular pathways mediated by “Ca. L. asiaticus” inoculated 3-year-old jiaogan seedlings. Each of them was graft-inoculated with one sweet orange scions with or without “Ca. L. asiaticus” in Dectember, 2009. RNA samples from three mandarin trees infected with 'Candidatus Liberibacter asiaticus' and three uninfected trees were used for affymatrix genochip
Project description:Huanglongbing (HLB) is a highly destructive, fast-spreading disease of citrus, causing substantial economic losses to the citrus industry worldwide. Nutrient levels and their cellular distribution patterns in stems and leaves of grapefruit were analysed after graft-inoculation with lemon scions containing 'Candidatus Liberibacter asiaticus' (Las), the heat-tolerant Asian type of the HLB bacterium. After 12 months, affected plants showed typical HLB symptoms and significantly reduced Zn concentrations in leaves. Micro-XRF imaging of Zn and other nutrients showed that preferential localization of Zn to phloem tissues was observed in the stems and leaves collected from healthy grapefruit plants, but was absent from HLB-affected samples. Quantitative analysis by using standard references revealed that Zn concentration in the phloem of veins in healthy leaves was more than 10 times higher than that in HLB-affected leaves. No significant variation was observed in the distribution patterns of other elements such as Ca in stems and leaves of grapefruit plants with or without graft-inoculation of infected lemon scions. These results suggest that reduced phloem transport of Zn is an important factor contributing to HLB-induced Zn deficiency in grapefruit. Our report provides the first in situ, cellular level visualization of elemental variations within the tissues of HLB-affected citrus.