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Phloem sap proteome of Citrus xlimon and Citrus xsinensis plants response to Trioza erytreae infestation

ABSTRACT: Trioza erytreae is a psyllid that feeds on citrus plants and other Rutaceae phloem sap, it is also a vector of the Huanglongbing disease (HLB) a highly aggressive disease caused by the bacteria Candidatus Liberibacter. One of the preferred plant hosts of Trioza erytreae is lemon (Citrus xlimon) plants, where it develops better than in other hosts. The reasoning behind this preference and better development is yet to be understood. To better understand this plant-insect interaction, two citrus hosts where infested with Trioza erytreae and their phloem sap proteomes were compared to their respective control. The hosts used in this study were the preferred host Citrus xlimon (lemon) and a common host Citrus sinensis (orange). The phloem sap was extracted from infested leaves of each host, and from leaves with a similar size and development from the respective controls. Protein extraction was preformed, and proteomic analysis was performed via nanoscale liquid chromatography coupled to tandem mass spectrometry (nanoLC-MS/MS). Four biological replicates were considered for each of the four treatments (infested lemon plants, control lemon plants, infested orange plants, control orange plants).

INSTRUMENT(S):

ORGANISM(S): Citrus Sinensis (sweet Orange) (citrus Aurantium Var. Sinensis) Citrus Limon

TISSUE(S): Phloem

SUBMITTER: Tomás Thormann Abranches de Magalhães

LAB HEAD: Natália Tomás Marques

PROVIDER: PXD043528 | Pride | 2026-03-02

REPOSITORIES: Pride

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Project description:Small RNAs (21-24 nt) are pivotal regulators of gene expression that guide both transcriptional and post-transcriptional silencing mechanisms in diverse eukaryotes, including most if not all plants. MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are the two major types, both of which have a demonstrated and important role in plant development, stress responses and pathogen resistance. In this work, we used a deep sequencing approach (Sequencing-By-Synthesis, or SBS) to develop sequence resources of small RNAs from Cucurbita maxima tissues (including leaves, flowers and phloem sap). The high depth of the resulting datasets enabled us to examine in detail critical small RNA features as size distribution, tissue-specific regulation and sequence conservation between different organs in this species. We also developed database resources and a dedicated website (http://smallrna.udel.edu/) with computational tools for allowing other users to identify new miRNAs or siRNAs involved in specific regulatory pathways, verify the degree of conservation of these sequences in other plant species and map small RNAs on genes or larger regions of the maize genome under study. Small RNA libraries were derived from leaves, flowers and phloem sap of Cucurbita maxima, variety Big Max. Total RNA was isolated using the TriReagent (Molecular Research Center) for leaves and phloem sap and the Plant RNA Purification Reagent (Invitrogen) for flowers, and submitted to Illumina (Hayward, CA, http://www.illumina.com) for small RNA library construction using approaches described in (Lu et al., 2007) with minor modifications. The small RNA libraries were sequenced with the Sequencing-By-Synthesis (SBS) technology by Illumina. PERL scripts were designed to remove the adapter sequences and determine the abundance of each distinct small RNA. We thank Pamela Green for providing the plant material as well as Kan Nobuta and Gayathri Mahalingam for assistance with the computational methods.

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.

			Action	DRS
	SDRF_PhloemOrangeLemon_StressTriozaErytreae.txt	Txt
	TM1_1.mzML	Mzml
	TM1_1.mzid.gz	Mzid
	TM1_1.raw	Raw
	TM1_2.mzML	Mzml

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Phloem sap proteome of Citrus xlimon and Citrus xsinensis plants response to Trioza erytreae infestation

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