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Role of long non-coding RNA in regulatory network response to ‘Candidatus Liberibacter asiaticus’ in citrus

ABSTRACT: The goals of this study are to reveal the transcriptional and regulatory dynamics of the lncRNAs in response to CLas infection and provide a reference for a better understanding of the role of lncRNAs involved in citrus HLB regulation. For the purpose, we obtained RNA-seq-based differential expression profile of the leave midrib of CLas- and mock-inoculated rough lemon and sweet orange at week 0, 7, 17, and 34 following infections with three biological replicates in the greenhouse. Approximately 2,523 million paired-end reads from 48 sample libraries were produced using Illumina HiSeq 2000. we systematically identified lncRNAs from rough lemon and sweet orange at four different time points of a greenhouse experiment and characterized their genomic transcriptional and regulatory dynamics. We predicted their potential regulatory genes and functions and constructed a co-expression network. Our study provides precious information and expands the knowledge of citrus lncRNA against HLB disease.

ORGANISM(S): Citrus sinensis Citrus jambhiri

PROVIDER: GSE215306 | GEO | 2023/03/15

REPOSITORIES: GEO

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Project description:Huanglongbing (HLB) is a worldwide devastating disease of citrus. There are no effective control measures for this newly emerging but century-old disease. A powerful oligonucleotide microarray of high-density 16S rRNA genes, the PhyloChip microarray, has been developed and effectively used to study bacterial diversity, especially from environmental samples. In this article, we aim to decipher the bacterial microbiome in HLB-affected citrus versus non-infected citrus as well as in citrus plants treated with ampicillin and gentamicin using PhyloChip-based metagenomics. The antibiotic treatments were conducted on the randomized complete block design with three replicates. For each replicate, 15 scions were treated in each antibiotic treatment (Amp and Gm) and control (CK1 and CK2). HLB-affected budsticks were sampled from severely HLB-affected field rough lemons (cv. Lemon #76) at the USDA-ARS-USHRL farm in Fort Pierce, FL and tested positive for Las by real-time qPCR. They were soaked in the antibiotic treatments; ampicillin sodium at a concentration of 1.0 g/L (Amp, Sigma-Aldrich, St. Louis, MO) or gentamicin sulfate at a concentration of 100 mg/L (Gm, Sigma-Aldrich, St. Louis, MO) and water as the diseased control (CK1), overnight in a fume hood under ventilation and lighting. Las-free budsticks, which tested negative by qPCR from healthy rough lemons, were also soaked in water as the healthy control (CK2). The budsticks were grafted onto two-year-old healthy grapefruit (Citrus paradisi 'Duncan') rootstocks and covered using plastic tape for three weeks. To improve scion growth, new flush from the rootstocks was removed after grafting and then allowed to grow. All experimental plants were grown in an insect-proof greenhouse. The first leaf samples from scions (rough lemon) and rootstocks (grapefruit) for DNA extraction were taken four months after inoculation, and second samplings were taken at six month after inoculation. The leaves were washed in tap water and then rinsed three times with sterile water. The midribs of the leaves were excised, frozen in liquid nitrogen, and stored at -80M-BM-0C. The midribs of five leaves from each sample were pooled, and DNA was isolated for qPCR analysis for Las bacterium. DNA from the leaf midribs of scions for the PhyloChipT G3 analysis, which was extracted from all samples of the same treatment, was pooled in equal amounts and quantified by the PicoGreenM-BM-. method. The PhyloChipTM G3 analysis was conducted by Second Genome Inc. (San Francisco, CA).

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.

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Role of long non-coding RNA in regulatory network response to ‘Candidatus Liberibacter asiaticus’ in citrus

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