Dataset Information

Huanglongbing Citrus Sinensis Phospho and Total Proteomics Data

ABSTRACT: Huanglongbing (HLB or Citrus Greening Disease) is a destructive citrus disease that has impacted the Florida citrus industry as well as global citrus industry for many years. There is yet to be a practical cure for HLB. Insecticide use targeting the psyllid vector and the burning, destruction and removal of all infected plants are the best solutions as of now. However, this is only a temporary and costly fix. The damage caused by HLB itself, the destruction of infected trees, and indirect effects in other economic sectors costs the Florida economy approximately $900 million per year. There are also problems with insecticide use; the high dosage needed the adequately suppress the transmission of the disease would contaminate the environment, rendering this solution unsustainable. Alternative solutions are needed. In the hopes of uncovering an alternative solution, we investigated the protein and phosphoprotein differences between healthy and diseased citrus plants to identify potential biomarkers essential towards fighting HLB within the trees themselves. For our experimental procedure, we grinded 2 grams of fresh leaf weight from the species Citrus sinensis (sweet orange). Phenol extraction was used to extract the proteins from four biological replicates of the control (healthy) leaves and four biological replicates of the treatment (diseased) leaves. Utilizing a Bradford assay and a 1-D gel electrophoresis, we evaluated the quality and quantity of the samples. Equal amounts of protein from the eight samples were digested with trypsin for 16 hours. Afterwards, we split each sample into two groups: one for phospho-enrichment and the other for total analysis. Phosphopeptides were enriched with the NuTip enrichment procedure for analysis of the phosphopeptides. Total peptides were cleaned up using ZipTip solid phase extraction. We ran the phosphopeptides and total peptides on an LC-MS/MS system, and the raw data files were processed with the Proteome Discoverer software using the citrus database downloaded from NCBI.

INSTRUMENT(S): Orbitrap Fusion ETD, Orbitrap Fusion

ORGANISM(S): Citrus Sinensis (ncbitaxon:2711)

SUBMITTER: Daniel Chen Sixue Chen

PROVIDER: MSV000084179 | MassIVE | Wed Aug 07 17:21:00 BST 2019

SECONDARY ACCESSION(S): PXD014934

REPOSITORIES: MassIVE

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Project description:Citrus greening or huanglongbing (HLB) disease is caused by Candidatus liberibacter asiaticus (CLas) and associated with an increase in pre-harvest fruit drop, for which the molecular mechanisms remain unknown. In order to understand the molecular basis of the HLB-associated fruit abscission, by means of RNA-Sequencing analysis (RNA-Seq), transcriptomes in citrus calyx abscission zones were analyzed and compared among fruit dropped (D) or retained (R) from healthy (h) or HLB-diseased (d) trees upon shaking the trees. Cluster analysis based on the transcript reads indicates that dropped fruit from HLB-diseased trees (Dd) have largest distances from all other groups. Differentially expressed genes (DEGs) were identified between Dd and Rd, Dh and Rh, Rd and Rh, Dd and Dh. Wilcoxon test of the whole dataset of DEGs revealed that consistently up-regulated genes in Dd versus Rd and Dd versus Dh are in the functional categories of “secondary metabolism, lipid metabolism and hormone-ethylene and –jasmonate”; while those down-regulated genes didn’t show clear pattern of regulation. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses indicated that the significant biological processes or pathways involved in HLB-related fruit abscission were those related to defense response, secondary metabolism and hormone signaling. Among them, “response to chitin” was the most significant (p= 9.95E-13) biological process, and “jasmonic acid biosynthesis” was the most significant (p= 4.6E-5) pathway. Genes related to synthesis and signaling of ethylene (ET) and jasmonate (JA) were consistently up-regulated, while abscisic acid, auxin, brassinosteroid, cytokinin, and gibberellin were generally down-regulated in Dd; but not in Dh. Consistent with the transcriptomic data, fruit ethylene production was detected in two third of the Dd fruit, but none of Rd, Dh or Rh fruit. And, in agreement with the hormone expression profiles, substantial numbers of downstream JA/ET-responsive defense related genes were up-regulated in Dd, but not in Dh. Thirty representative DEGs covering categories of hormone, secondary metabolism, and JA/ET responsive defense responses were verified by qRT-PCR. The results indicate that HLB-associated pre-harvest fruit abscission is mediated by JA/ET signaling, which has been known to be triggered by infection of necrotrophic pathogens.

Project description:To evaluate the transcriptome response of roots towards Hoagland solution of two-year-old healthy (C) and HLB-affected (T) sweet orange (Citrus sinensis L.) cultivar Midsweet grafted on Kuharskei Carrizo rootstock at three different time points; D1 (at the start of the experiment), D2 (After 3 days of Hoagland solution), D3 (After nine days of Hoagland solution), were performed using RNA sequencing analysis. A total of 9, 19, and 2324 DEGs were expressed in HLB-affected and healthy trees feeder roots on D1, D2, and D3, respectively. Due to a small number of DEGs in HLB-affected and healthy trees roots on D1 and D2, enrichment analysis could not be performed. At D3, Gene Ontology (GO) enrichment analysis was performed using upregulated and downregulated DEGs in HLB-affected roots compared to healthy roots. For the upregulated DEGs in HLB-affected roots, the most enriched biological GO categories were related to transport, cellular amino acid metabolic process, oxoacid metabolic process, organic acid metabolic processes, phenylpropanoid biosynthesis process, response to carbohydrate stimulus, ethylene and the jasmonic acid-mediated signaling pathway, and regulation of plant hormone. The biological GO categories based on the number of DEGs associated with downregulated DEGs were the developmental process, phosphate metabolic process, protein modification process, defense response, growth, cell cycle, cell death, and ABA-mediated signaling pathway. The results of this study strongly suggest; although, the reduced biomass limits the nutrient uptake capacity in the plants as a whole, so in order to compensate for reduced root to shoot ratio the existing root undergo anatomical and transcriptomic changes to improve nutrient uptake efficiency to meet the nutrient demand of shoot systems. It is likely that higher inputs of energy in nutrient uptake possibly results in reducing the root longevity of HLB-affected trees. Good nutrition management practices are critical for the survival of HLB-affected trees as the availability and uptake of nutrients allow HLB-affected trees in response to abiotic and biotic stress.

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Huanglongbing Citrus Sinensis Phospho and Total Proteomics Data

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