Endosymbiont hunting in the metagenome of Asian citrus psyllid (Diaphorina citri)

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The Asian citrus psyllid (D. citri Kuwayama or ACP) is host to 7+ bacterial endosymbionts and is the insect vector of Ca. liberibacter asiaticus (Las), causal agent of citrus greening. To gain a better understanding of endosymbiont and pathogen ecology and develop improved detection strategies for Las, DNA from D. citri was sequenced to 108X coverage. Initial analyses have focused on Wolbachia, an alpha-proteobacterial primary endosymbiont typically found in the reproductive tissues of ACP and other arthropods. The metagenomic sequences were mined for wACP reads using BLAST and 4 sequenced Wolbachia genomes as bait. Putative wACP reads were then assembled using Velvet and MIRA3 assemblers over a range of parameter settings. The resulting wACP contigs were annotated using the RAST pipeline and compared to Wolbachia endosymbiont of Culex quinquefasciatus (wPip). MIRA3 was able to reconstruct a majority of the wPip CDS regions and was selected for scaffolding with Minimus2, SSPACE and SOPRA using large insert mate-pair libraries. The wACP scaffolds were compared to wPip using Abacas and Mauve contig mover to orient and order the contigs. The functional annotation of scaffolds was evaluated by comparing it to wPip genome using RAST. The draft assembly was verified using an OrthoMCL based comparison to the 4 sequenced Wolbachia genomes. We expanded the scope of endosymbiont characterization beyond wACP using 16S rDNA and partial 23S rDNA analysis as a guide. Results will be presented regarding endosymbionts, their potential interactions and their impact on the disease of citrus greening.

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Endosymbiont hunting in the metagenome of Asian citrus psyllid (Diaphorina citri)

  1. 1. Endosymbiont hunting in themetagenome of Asian citrus psyllid(Diaphorina citri)Surya Saha1, Wayne B. Hunter 2 andMagdalen Lindeberg 1Magdalen Lindeberg1 Department of Plant Pathology and Plant-Microbe Biology, CornellUniversity, Ithaca, NY, USA, 2 USDA-ARS, US Horticultural Research Lab, FortPeirce, FL, USASFAF 2012, Santa Fe
  2. 2. HLB found in Florida2005Worldwide distribution of CitrusGreening/Huanglongbing1st scientificreport of HLB inChina – 1919SFAF 2012, Santa Fe
  3. 3. What is the impact of this disease?Citrus – a 9.3 billion dollar industry in Florida and 1.2 billion dollars in California• Leave agricultural practices unchanged - significant loss of production• Begin expensive , labor intensive controls - increase cost of productionhealthy infectedIncreased cost of citrus products for consumersSFAF 2012, Santa Fe
  4. 4. What is known so far?The causal agent:How it spreads:All evidence points to the bacteriumCa. Liberibacter asiaticusVectored by Asian citrus psyllid (ACP)How to control it:Kill the pathogenLimit spread of the insectLimit the spread of infected plantsResistant host plantsComplicated by unusual biologySFAF 2012, Santa Fe
  5. 5. The causal agent:Ca. Liberibacter asiaticus• 1.2 Mb reduced genome• Florida strain sequencedCa. Liberibacter asiaticus in citrus phloem cellsComplications:• Cannot be cultured making experimental characterization verydifficult• Until recently was on the select agent list adding to the difficultiesof experimental characterization• How it causes disease is not clearSFAF 2012, Santa Fe
  6. 6. How it gets around:Ca. Liberibacter asiaticus is vectored byAsian Citrus PsyllidInsect transmission greatly enhancesInsect transmission greatly enhancespotential for rapid long distance spreadPsyllid feeds on many members of the citrus familyOnce infected, each plant becomes a disease reservoir fromwhich uninfected psyllids can pick up bacteriaSFAF 2012, Santa Fe
  7. 7. Overview• Citrus Greening website• Ca. Liberibacter asiaticus annotation• Asian citrus psyllid (ACP)• Genome assembly• Transcriptome mining for transgenic plants• Transcriptome mining for transgenic plants• Host-endophyte metagenomics• Symbionts of ACP• Endophytic populations of Maize• Pseudomonas syringae pathogen genomics• T1-like strain• Pseudomonas viridiflavaSFAF 2012, Santa Fe
  8. 8. Citrus Greening websiteCommunity resourceGenome browserGenome annotationTranscription factors andbinding sitesSubcellular localization ofproteinsRpoH(heat)RirA(iron)RpoD(primary)SFAF 2012, Santa Fe
  9. 9. Price 2007ACP gene hunting for transgenic plantsDistrupt the osmotic balance of phloemfeeding HemipteransMining for osmoregulatory genes in ACPTargeted denovo transcriptome Price 2007assemblyPotato psyllidWhite flyPeach-potato aphidRNAi-mediated knockdown of osmoregulatory genesAngela DouglasSFAF 2012, Santa Fe
  10. 10. ACP genome assemblySample from FloridaIllumina sequencingPaired end reads (37 Gb)Mate-pair read libraries2k (8.7 Gb)5k (7.5 Gb)psyllid.org5k (7.5 Gb)10k (7.1 Gb)~400 Mb genome180X coverageDraft assembly analysisGene findingRepeat identificationSecond round of sequencing with Pacificbiosciences underwayWayne HunterCliff HanSFAF 2012, Santa Fe
  11. 11. LiberibacterBig picture for molecular interactionsCould lead to identification ofmetabolically complementarybacteria for co-culturing withLiberibacterpsyllid citrusSFAF 2012, Santa Fe
  12. 12. Why endosymbionts?Significant impact on diverse hostprocessesNutritional statusReproductionLifespanResistance to insecticidesMeans to explore population dynamicsand relatedness to other isolatesand relatedness to other isolatesKnown examples of highly-evolvedhost-symbiont relationshipBuchnera and AphidsPsyllid microbiome diversity10 bacteria reported in ACP (Florida)5 additional bacteria reported inclosely related potato psyllid (B.cockerelli)Buchnera-derived essentialamino acids supporting thegrowth of pea aphid larvae.[ Gündüz and Douglas 2009]SFAF 2012, Santa Fe
  13. 13. Candidate endosymbiontsDescription Length (bp) CoverageLength(bp)Klebsiella variicola At-22 chromosome, completegenome 5,458,505 1.144% 307,111Salmonella enterica subsp. enterica serovar Typhi str.Ty2 chromosome, complete genome 4,791,961 1.266% 464,747Staphylococcus epidermidis ATCC 12228chromosome, complete genome 2,499,279 3.077% 60,415Acidovorax avenae subsp. avenae ATCC 19860Why Wolbachia?Known obligate association with up to 16% of insectsspeciesPositive correlation of titer with Ca. liberibacer asiaticusAcidovorax avenae subsp. avenae ATCC 19860chromosome, complete genome 5,482,170 0.473% 71,218Acinetobacter sp. DR1 chromosome,completegenome 4,152,543 2.763% 102,446Herbaspirillum seropedicae SmR1 chromosome,complete genome 5,513,887 0.949% 23,509Wolbachia endosymbiont of Culex quinquefasciatusPel, complete genome 1,482,455 86.787% 1,140,899Methylibium petroleiphilum PM1 chromosome,complete genome 4,044,195 0.318% 20,107Positive correlation of titer with Ca. liberibacer asiaticusAssociated with Cytoplasmic Incompatibility in hostsUsed for vector control in dengue feverSFAF 2012, Santa Fe
  14. 14. Read capture using baitsWolbachia endosymbiont of Drosophila melanogasterWolbachia endosymbiont of Culex quinquefasciatus Pel (wPip)Wolbachia sp. wRiWolbachia endosymbiont strain TRS of Brugia malayiDataset Total (bp) 100% 90% 85%SFAF 2012, Santa FePaired-end 33,708,218,600 127,335,000 228,893,000 237,906,000Mate-pair 2k 7,020,642,800 11,200 45,200 63,000Mate-pair 5k 6,000,789,600 10,000 43,000 66,800Mate-pair 10k 7,307,423,600 2,895,000 4,427,600 4,909,000Results of mining putative wACP reads from the ACP metagenome
  15. 15. Assembler Contigs N50Contigs >1kLength ofContigs >40kVelvetOptimizer(n50, 83 mer)111 79935 53 807171VelvetOptimizer(n50/tbp, 95 mer)118 84957 25 744357MIRA3 167 23068 105 247976Annotation0 1000 2000Total CDSRNAFunctional - shared with wPipFunctional - wPip aloneFunctional - not in wPipwPipMIRA3VelvetOptimizer (n50/tbp, 95 mer)VelvetOptimizer (n50, 83 mer)WorkflowVelvetOptimizer (n50, 83 mer)ScaffoldingDescription Contigs Longest N50Contigs >1kSOPRA / wACP2 101 89608 41594 77Minimus2 /wACP199 106898 41594 76Minimus2 /wACP3104 206691 52418 66wACP1 wACP2 wACP3Total CDS 1176 1190 1183RNA 36 36 36Functional - sharedwith wPip608 610 608Functional - wPipalone24 23 24Functional - not inwPip23 22 23Functionalannotationusing RASTSFAF 2012, Santa Fe
  16. 16. Arrangingscaffoldsusing wPip asreferenceFunctionalsubsystemsidentified inwACP usingRASTSFAF 2012, Santa Fe
  17. 17. Wolbachia wPip endosymbiont of Culex quinquefasciatus PelWolbachia sp. wRiWolbachia endosymbiont strain TRS of Brugia malayi0200400600800100012001400Total proteinsCoreCoreOrthoMCL analysis of pan-proteomeCore / Core paralogousShared / Shared paralogousLineage specific660 core protein clusters per Wolbachia genome659 core protein cluster representatives found in wACPMissing core cluster consists entirely of hypotheticals32 lineage specific genes in wACP w.r.t. wPipAll have unknown function11 have homologs in other endosymbionts of mosquitoWolbachia endosymbiont of Drosophila melanogasterCoreparalogousSharedSharedparalogous Lineagespecific LineagespecificparalogousSFAF 2012, Santa Fe
  18. 18. Ankyrin domain proteinsTandemly arranged 33 residue repeatsSufficiently divergent at nucleotide level to allow assemblyCandidate for host protein manipulationwACP ankyrin repetoire54 predicted proteins contain ankyrin domain34 have close homologs in wPippk2 group of ankyrin proteins presentpk2 group of ankyrin proteins presentCytoplasmic incompatibility in CulexFeminization in isopodsType IV secretion system2 cluster arrangement of Type IV genesWidely conserved in Wolbachia and present in wACPSFAF 2012, Santa Fe
  19. 19. Comparative phylogeny of wACPUSA populationsformed a separateclade from fourclade from fourwACP populationsfrom China.SFAF 2012, Santa Fe
  20. 20. ConclusionsTargeted genome reconstruction strategy forendosymbiontsRead mapping approach for candidate endophytesRecommendationsMIRA3 produced more fragmented but qualitativelyMIRA3 produced more fragmented but qualitativelybetter assemblies than VelvetSOPRA and SSPACE are effective for scaffolding whencoupled with Minimus2CaveatsMissing lineage specific regionsLow throughput methodSFAF 2012, Santa Fe
  21. 21. AcknowledgementsMagdalen LindebergCornell UniversityDave SchneiderUSDA-ARS, IthacaCitrus greening / Wolbachia (wACP)Asian Citrus Psyllid genome• Wayne Hunter• Cliff Han• Goutam Gupta• Olga Chertkov• Justin Reese• Brandi CantarelAsian Citrus Psyllid transcriptomicsPseudomonas pathogens• Alan CollmerFundingMeta-Maize• Rebecca Nelson• Alice Churchill• Santiago Mideros• Laura Morales• Angela Douglas• Xiangfeng Jing• Alan Collmer• Greg Martin• Chris Smart• Lisa Jones
  22. 22. Questions??Questions??

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