Citrus response to Candidatus Liberibacter asiaticus and its vector Diaphorina citri. Nabil Killiny Assistant professor University of Florida

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1 Citrus response to Candidatus Liberibacter asiaticus and its vector Diaphorina citri Nabil Killiny Assistant professor University of Florida

2 Lecture content Huanglongbing (HLB) caused by candiditus Liberibacter asiaticus (CLas) and transmitted by Asian Citrus Psyllid Role of CLas cell-to-cell signaling system in the interaction between the bacterial pathogen with its plant host and the insect vector. Protein-protein interactions between Clas and psyllid Importance of ATP translocase in CLas growth in their host. Phloem sap, haemolymph, and honeydew chemical composition.

3 Citrus Greening Huanglongbing (HLB) seriously threatens the citrus industry HLB symptoms include1- Yellow shoots yellow dragon, and blotchy mottle leaves. The causal agent is Candidatus Liberibacter asiaticus bacteria (CLas) Asian citrus psyllid (ACP), Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is the vector of CLas is phloem restricted and ACP is phloem sap esources/pages/iw00006.aspx feeder.

4 Insect transmission of plant pathogens Mechanisms of transmission Persistence circulation propagation VECTOR FEEDING: MECHANISMS AND BEHAVIOR Host Vector Acquisition Inoculation Pathogen

5 Molecular and Biochemical Interactions Pathogen-host interactions Vector to host transition Vector-host interactions Host to vector transition Pathogen-vector interactions

6 CLas Transmission Circulative Propagative Bacteria pass through the biological barriers (Gut and salivary glands)! Specific interactions (receptorligand) Biofilm formation Bacterial multiplication in haemolymph! Nutrition (sugars, Amino acids,. ) Quorum sensing! Two components system

7 Biofilm formation on the gut surface Michael Davis, CREC Lazdunski et al 2004 Nature Clas Colonization of ACP

8 LuxR in CLas genome but not LuxI V f Pa LAS M t Ec At Pf Bs Cf M LuxR kda M 850 nts 741 nts 650 nts LuxR 34 kda Amplification of LuxR gene of HLB from total nucleic acids isolated from HLB infected citrus

9 Induction of fluorescence of Clas-luxR/GFP transformed E. coli by different AHLs Method according to Kock et al 2005 Plate is overlaid with indicator bacterium (biosensor) A. Tumefaciens (pahl-ice) provided by Lindow lab, Berkeley, UC

10 AHL-producing and non-ahl-producing bacteria S. Subramoni and V. Venturi 2009

11 Hypothesis Insect Host Plant Host The complex trigger the growth and biofilm formation The complex trigger the multiplication and planktonic status

12 Expression of LuxR in Citrus using CTV-Based vector R² = Infection rate LuxR- LuxR+ Linear (LuxR- ) R² = weeks

13 Figure 8: Pathogenicity of CLas in LuxR expressedcitrus.

14 On going work Expressing luxi (AHL) from three bacteria in citrus phloem sap. Table showed the genes to be tested. In red, genes are already expressed in citrus Localized symptoms were noticed for the three tested genes. In the yellow branches Ct is much lower than the rest of tree indicating deficiency in bacterial moving Organism AHL structure Gene Accession name no. Aeromonas hydrophilia Agrobacterium vitis Agrobacterium tumefaciens N-(butyryl)-L- homoserine lactone (BHL), N-(hexanoyl)- L-homoserine lactone (HHL)(Swift et al., 1997) Producing long-chain AHLs, including C16-HSL, 3-oxo-C16- HSL, C16:1-HSL, and 3-oxo-C16:1-HSL (Subramoni & Venturi, 2009, Savka et al., 2011, Hao & Burr, 2006) N-(B-oxo-octan-1-oyl)-L-homoserine lactone(hwang et al., 1994) AhyI AvsI CAA61653 DQ TraI L Burkholderia cepacia N-octanoyl-HSL(Lewenza et al., 1999) CepI AF Chromobacterium HHL(Oulmassov et al., 2005) CviI AY violaceum 1 Panteoa 3-O-C6-HL (Watson et al., 2002) EsaI stewartii formerly L32183 Erwinia stewartii Pectobacterium N-3-oxohexanoyl-L-homoserine ExpI carotovorum lactone (Brader et al., 2005) AY Pseudomonas aeruginosa Pseudomonas aeruginosa Pseudomonas fluorescens Rhizobium leguminosarum bv. viciae Rhizobium leguminosarum bv. viciae Rhizobium leguminosarum bv. viciae Rhodobacter sphaeroides Serratia liquifaciens Sinorhizobium meliloti Vibrio anguillarum 3-Oxo-C12-HSL, 3-oxo-C10-HSL (Subramoni & Venturi, 2009) LasI 1 AHW (Protein ID) C4-HSL (Subramoni & Venturi, 2009) RhlI U Six acyl-hsls, including the 3-hydroxy forms, N-(3-hydroxy-hexanoyl)-Lhomoserine lactone(3-oh-c6-hsl), N-(3- hydroxy-octanoyl)-l-homoserine lactone (3-OH-C8-HSL), and N-(3-hydroxydecanoyl)-L-homoserine lactone (3-OH- C10-HSL); the alkanoyl forms hexanoylhomoserine lactone (C6-HSL) and octanoyl-homoserine lactone (C8- HSL)(Khan et al., 2005) 3-OH-C14:1-HSL (Wilkinson et al., 2002, Edwards et al., 2009) C6-HSL, C7-HSL, and C8-HSL (Wilkinson et al., 2002) N-(3-hydroxyoctanoyl)-L-homoserine lactone 3-OH-C8-HSL (Edwards et al., 2009) 7,8-cis-N-(tetradecanoyl)-HSL(Puskas et al., 1997) N-(butyryl)-L- homoserine lactone (BHL) (Oulmassov et al., 2005) Producing several long-chain AHLs ranging from C12- to C18-HSL, including C14-HSL, 3-oxo-C14-HSL, C16 : 1-HSL, 3- oxo-c16-hsl, C18-HSL(Subramoni & Venturi, 2009) N-(oxodecanoyl)-L-homoserine lactone (ODHL)(Oulmassov et al., 2005) PhzI CinI RhiI RaiI CerI SwrI SinI VanI Vibrio fischeri OHHL (Schaefer et al., 1996) LuxI AAC AF CAK10388 (Protein ID) AJ AF U CAC (Protein ID) U69677 AAD (Protein Vibrio harveyi Yersinia enterocolitica Yersinia pseudotuberculosi s N-(hydroxybutyryl)-L- homoserine lactone (HBHL)(Oulmassov et al., 2005) OHHL and HHL(Fray et al., 1999) N-(3-oxohexanoyl)-HSL(Atkinson et al., 1999) ID) LuxM L13940 Yenl X YpsI AF

15 Conclusion: Clas genome possess luxr but not luxi (LuxR sol system) LuxR is functional and responds to signals from psyllid and citrus. Expressing LuxR protein in citrus phloem sap enhances CLas pathogenicity and increases its mobility (binding to citrus signals affecting the bacterial attachment and increase the movement) Expressing AHLs in citrus phloem sap inhibit Clas pathogenicity by reducing the movement (cells are more attached and biofilm enhanced)

16 CLas Transmission Circulative Propagative Bacteria pass through the biological barriers (Gut and salivary glands)! Specific interactions (receptorligand) Biofilm formation

17 Circulative propagative transmission Mollicutes Spiroplasmas Phytoplasmas

18 ACP receptors and CLas membrane proteins 3 ph 10 Detection of insect proteins

19 E E E Insect total proteins Gut proteins

20 Insect total proteins 1-ATP synthase alpha subunit 2-ATP synthase beta subunit 3- Actin 4-beta-tubulin 5- alpha-tubulin 6-Transitional endoplasmic reticulum ATPase TER kda glucose-regulated protein, 8-arginine kinase. LC-MS-MS

21 CLas Transmission Circulative Propagative Bacterial multiplication in haemolymph! Nutrition (sugars, Amino acids,. ) Biofilm formation ATP!!!

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23 Absorbance at 260 nm UK 1 UK 2 NAD UK 3 CMP UK 4 UK 5 AMP NAD CMP AMP UK 6 NAD P NAD P CDP UMP UMP CTP UDP-GalNAc ADP UDP- UDP-Glc Gal GMP CTP UDP-GalNAc ADP GMP IMP IMP UDP UDP ATP GDP-Fuc GDP-Man GDP ITP GTP ATP GDP-Fuc GDP-Man UTP GDP IDP FAD GTP ITP Time (min) Nucleotide μg/insect) CLas- Clas+

24 * * ATP / insect (ng) ATPase/GTPase / insect (μunit) Enzymatic quantification

25 * 10 Nucleotides ratio * Fold change ATPase ATP synthase alpha ATP syntase beta Gene expression AMP/ATP ADP/ATP

26 CLas Transmission Circulative Propagative Biofilm formation Bacterial multiplication in haemolymph! Phloem sap! Nutrition AHL and LuxR binding compound Break 10 min

27 Phloem sap composition. LuxR-binding compounds Haemolymph composition AHL Honeydew composition medium to culture CLas artificial diet solution for ACP

28 Chemistry of phloem sap, haemolymph, and honeydew Amino acids, sugars, organic acids, and minerals Chemistry ACP hemolymph ph Brix-value Total amino acids butanedioic acid 0.20% L-proline 0.62% Chemistry ACP honeydew malic acid 3.00% D-turanose 0.15% trehalose 20.99% ribitol 0.01% citric acid quinic acid 0.56% 0.03% Gluco D-fructose % se % D-fructose % glucose % mannitol 0.01% myo-inositol % L-proline 0.04% butanedioic acid 0.00% trehalose 14.04% D-turanose 0.01% malic acid 0.07% quinic acid 0.05% D-fructose citric acid 0.00% Glucose % D-fructose % % glucose % mannitol 0.02% myoinositol 1 myoinositol 0.09% % inositol 0.08% sucrose 52.64% myo-inositol % sucrose 71.52% ACP-Honeydew collected from healthy pineapple inositol 1.05% ACP-Honeydew collected from CLas infected pineapple Chemistry of Citrus phloem sap Clas susceptibl e varieties Clas tolerant varieties ACP preferable varieties Clas Tolerant and ACP preferable varieties ACP Nonpreferable varieties

29 Phloem sap collection The Stylectomy method cut the insect stylet while aphid is feeds on the host plant and the exudate is collected using microcapillary. Pure phloem sap The amount obtained by is small The incision method Spontaneous bleeding of the phloem sap. It does not work with most plants because the rapid accumulation of callose and P-protein in the sieve plates after incision stops the phloem bleeding The EDTA method Ethylenediaminetetraacetic acid (EDTA) Avoids accumulation of callose and P- protein in the phloem sieve. The tip of the petioles or a piece of bark tissue is immersed in EDTA solution for a few hours It does not measure the real concentration of the phloem sap components

30 I-Phloem sap collection II-Derivatization III-GC-MS Trimethylsilyl (TMS) Methyl chloroformate (MCF) Pure phloem sap EDTAenhanced exudates N-methyl-N- [tert-butyl dimethylsilyl]- trifluroacetamide (MTBSTFA)

31 Pure sap EDTA exudate MTBSTFA Percentage composition of amino acids in the phloem sap from pineapple sweet orange MCF

32 Pure sap EDTA exudate TMS Percentage composition of sugars in the phloem sap from pineapple sweet orange

33 Pure sap EDTA exudate TMS MCF Percentage composition of organic acids in the phloem sap from pineapple sweet orange MTBSTFA

34 Pure sap EDTA exudate Average Chemical composition of pineapple sweet orange expressed as percentage composition of the major groups (averages of the different derivatization methods)

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36 Palestine lime B PCA for citrus varieties using their phloem sap composition

37 Phenylalanine R² =

38 Phloem sap Haemolymph NK-HD % 0 NK-Hemo % 0 NK-PS Amino acids Organic acids Sugars ; Scan EI+ TIC 7.11e10 Scan EI+ TIC 7.11e10 Scan EI+ TIC Phloem sap Honeydew Macrophylla TMS 0.3 ul #5 NK-PS % 0 NK-HD % ; Feb :06: ; Scan EI+ TIC 4.74e10 Scan EI+ TIC 6.16e10 Time

39 P>0.05 P<0.05 Sucrose 20% Phloem sap mimic solution Lifecycle in artificial diet system! Transovarial transmission RNAi

40 Phloem sap chemical composition (AA, OA, FA, Sugars, minerals, nucleotides, sugar nucleotides) Proteome map of phloem sap Diet solution to rear ACP for RNAi and Identify LuxR binding compounds from phloem sap Isolation of AHL from haemolymph Culture CLas

41 CLas Biology Circulative Propagative Bacteria pass through the biological barriers (Gut and salivary glands)! Specific interactions (receptorligand) Bacterial multiplication in haemolymph and phloem sap! Nutrition (sugars, Amino acids, ATP,. ) Biofilm formation Quorum sensing! Two components system

42 Next Week Chemistry Varieties differences Citrus response to HLB (pathogen and/or vector) Volatiles Carboxylic compounds Phytohormones Pigments Non protein AA Genetic manipulation to enhance resistance RNAi in citrus RNAi in psyllid RNAi in psyllid mediated by citrus

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