Project description:Huanglongbing, or citrus greening disease, has devastated the citrus industry. It is associated with the gram negative bacterium Candidatus Liberibacter asiaticus (CLas) that can be transmitted by Diaphorina citri, the Asian citrus psyllid. For transmission to occur, CLas must cross the gut of the ACP to circulate through the insect body. The insect gut is the first site of widespread interactions between the CLas and the ACP and forms a barrier to transmission. To investigate the effect of CLas exposure on this dynamic interface, we performed RNAseq and mass spectrometry-based proteomics to analyze the transcriptome and proteome respectively of dissected ACP guts. We found changes in iron metabolism, insecticide resistance, immune system, and apoptosis. We identified 83 long non-coding RNAs that are responsive to CLas, two of which have no homology to other organisms in NCBI. We also determined that Wolbachia, a symbiont of the ACP, undergoes protein regulation when CLas is present. Fluorescent in situ hybridization (FISH) confirmed that Wolbachia and CLas can inhabit the same ACP gut cell, but do not co-localize. These data provide a snapshot of the ACP gut under normal and CLas-exposed conditions, and provide tools to better understand the insect vector of the citrus greening pathosystem.
Project description:‘Candidatus Liberibacter asiaticus’ (CLas), the bacterial pathogen associated with citrus greening disease, is transmitted by Diaphorina citri, the Asian citrus psyllid (ACP). We used quantitative mass spectrometry to compare the proteomes of CLas(+) and CLas(-) populations of D. citri. This experiment used adult mixed sex whole insect samples as starting material for protein extraction. DNA extracted from CLas(+) ACP was tested by qPCR to confirm presence of CLas.
Project description:‘Candidatus Liberibacter asiaticus’ (CLas), the bacterial pathogen associated with citrus greening disease, is transmitted by Diaphorina citri, the Asian citrus psyllid (ACP). Percoll gradient density centrifugation was used to fractionate an ACP homogenate to generate a sample enriched for intact microbial cells (CLas and insect endosymbionts) and associated ACP cells. Proteins were extracted from Percoll gradient fractions prepared in triplicate from CLas(-) ACP samples and CLas(+) ACP samples.
Project description:The Asian citrus psyllid (Diaphorina citri) is a pest of citrus and the primary insect vector of the bacterial pathogen, ‘Candidatus Liberibacter asiaticus’ (CLas), which is associated with citrus greening disease. Variability in CLas titer in insects collected from infected plants has been attributed in part to the host plant from which the insects were collected. CLas accumulates to high titers in infected Citrus macrophylla, and in D. citri feeding on the infected plants of this species. In contrast, in the citrus relative Murraya paniculata, CLas titers remain low in infected plants and in D. citri exposed to infected plants. In this study, top-down and bottom-up proteomics methods were used to investigate the impact of these different host plants on D. citri protein expression. Difference in gel electrophoresis (DIGE) was used to identify protein spots on two-dimensional gels that were larger in one of three insect sample classes compared to the other two: D. citri continuously reared on C. macrophylla, D. citri reared continuously on M. paniculata, and D. citri transferred to M. paniculata for five days feeding after continuous rearing on C. macrophylla. Peptide mass spectrometry was used to identify and quantify proteins in target spots upregulated in each sample class. Shotgun proteomics was used to identify and quantify proteins from analysis of tryptic peptide samples prepared from whole insects from four sample classes: the reciprocal host switch condition (D. citri transferred to C. macrophylla for five days feeding after continuous rearing on M. paniculata) in addition to the three sample classes used in DIGE analysis. Integration of the results of both analyses reveals proteins identified by separate experimental workflows to be upregulated in insects adapted to each host plant, and in insects adapting to a novel host plant. A peptidoglycan-degrading protein involved in the immune response to bacterial pathogens was found to be upregulated in M. paniculata-reared D. citri. In the absence of CLas infection, host plant factors specific to M. paniculata may prime the antibacterial immune response in D. citri. Understanding the insect proteins involved in the adaptation of D. citri to host plants with variation in their susceptibility to CLas will inform the development of control strategies aimed at stopping the spread of citrus greening disease.
Project description:To identify genes associated with citrus peel development and manifestation of peel disorders, we analyzed flavedo, albedo and juice sac tissues from navel orange displaying, and not displaying, the puff disorder. Symptomatic and healthy M-bM-^@M-^\NavelM-bM-^@M-^] orange fruits were harvested from an orchard located in in Pauma Valley, San Diego County, California, USA. Sampling for all analysis (healthy or disordered Navel orange) was performed at the same time, from trees grown under the same agronomic, soil, and environmental conditions. Healthy and disordered fruits were analyzed at the mature stage. All transcriptome analysis was performed on mature fruit. For each type of fruit, three tissues (flavedo, albedo, and juice sacs) from three different trees (biological replicates) were separately analyzed. Four symptomatic fruits comprised one biological replicate each. Two healthy fruits comprised two biological replicates of control samples. A 1 cm-thick equatorial disc and four sections (N, S, E, and W) were cut per fruit. Each section of flavedo, albedo, and juice sac tissue was dissected. gene expression variation underlying quality trait, different genotypes
Project description:Citrus variegated chlorosis (CVC), caused by Xylella fastidiosa, is an important citrus disease that produces chlorotic injuries on leaves and reduced fruit size. This bacterium colonizes plant xylem, thereby interrupting sap flow. Other disease symptoms depend on environmental factors, since asymptomatic and symptomatic CVC plants may be genetically similar. The endophytic microbiome comprises many microbial species that may interact with pathogens, reducing disease symptoms and improving plant growth. However, the genetic and physiological mechanisms that underlie this interaction are largely unknown. In this study, the citrus endophytic bacterium Methylobacterium mesophilicum SR1.6/6 was isolated from healthy plants. This bacterium was able to colonize citrus xylem and could be transferred from plant to plant by Bucephalogonia xanthopis (Insecta), suggesting that this endophytic bacterium may interact with X. fastidiosa in planta, as a result of co-transmission by the same insect vector. To better understand how X. fastidiosa genetic responds to the presence of M. mesophilicum in the same environment, we used microarrays to evaluate the transcriptional profile of X. fastidiosa, after in vitro co-cultivation with M. mesophilicum SR1.6/6. The results showed that during co-cultivation with M. mesophilicum, X. fastidiosa downregulated genes related to growth, while genes related to energy production (cellular respiration) and transport were upregulated. Moreover, X. fastidiosa modulates genes associated with molecular recognition, nutrient competition and the stress response, suggesting the existence of a specific adaptive response to the presence of M. mesophilicum in the culture medium
2014-04-18 | GSE56901 | GEO
Project description:Florida citrus insect pest RNA virome
Project description:Citrus disease resistance breeding has been advanced to introduce CTV resistance of trifoliate orange to citrus. Because the quality of the fruit of trifoliate ogate was low, backcross with citrus was necessary. In the case of citrus, it takes several years from flowering to obtaining next-generation seeds. Therefore, we generated transformants for the early flowering genes (citrus FLOWERING LOCUS T: CiFT) using CiFT co-expression vector construct and promoted generation. In Japan, it is difficult to plant transformants in the field. Therefore, it was decided to select null segregant lacking transgene from backcross progenies. In order to prove that the transgene has been completely removed, it is necessary to prove that no vector conract is present on the genome. Tthis matter was proved by CGH analysis.
Project description:Fruit ripening in Citrus is not well understood at the molecular level. Knowledge of the regulatory mechanism of citrus fruit ripening at the post-transcriptional level in particular is lacking. Here, we comparatively analyzed the miRNAs and their targeted genes in a spontaneous late-ripening mutant, ?Fengwan? sweet orange (MT) (Citrus sinensis L. Osbeck), and its wild-type counterpart ('Fengjie 72-1', WT). Using high-throughput sequencing of small RNAs and RNA degradome tags, we identified 107 known and 21 novel miRNAs, as well as 225 target genes. A total of 24 miRNAs (16 known miRNAs and 8 novel miRNAs) were shown to be differentially expressed between MT and WT. The expression pattern of several key miRNAs and their target genes during citrus fruit development and ripening stages was examined. Csi-miR156k, csi-miR159 and csi-miR166d suppressed specific transcription factors (GAMYBs, SPLs and ATHBs) that are supposed to be important regulators involved in citrus fruit development and ripening. In the present study, miRNA-mediated silencing of target genes was found under complicated and sensitive regulation in citrus fruit. The identification of miRNAs and their target genes provide new clues for future investigation of mechanisms that regulate citrus fruit ripening.