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Isolation of genes differentially expressed during the defense response of Cassava (Manihot esculenta Crantz) to whitefly (Aleurotrachelus socialis Bondar) attack
 

Isolation of genes differentially expressed during the defense response of Cassava (Manihot esculenta Crantz) to whitefly (Aleurotrachelus socialis Bondar) attack

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    Isolation of genes differentially expressed during the defense response of Cassava (Manihot esculenta Crantz) to whitefly (Aleurotrachelus socialis Bondar) attack Isolation of genes differentially expressed during the defense response of Cassava (Manihot esculenta Crantz) to whitefly (Aleurotrachelus socialis Bondar) attack Presentation Transcript

    • Isolation of genes differentially expressed during the defense response of Cassava (Manihot esculenta Crantz) to whitefly (Aleurotrachelus socialis Bondar) attack Adriana Bohórquez Chaux Diana Bernal Bernardo Arias Anthony Bellotti Joe Tohme
    • General Objective Understand the defense mechanisms of cassava (Manihot esculenta Crantz) at the gene expression level, in response to whitefly (Aleurotrachelus socialis Bondar) attack.
    • African Cassava Mosaic Disease CMD
    • Neotropics Whitefly: y Aleurotrachelus socialis Yield losses 5- 79%
    • Susceptible genotype Source: Cassava Entomology, CIAT
    • MEcu-72 Whitefly RESISTANT CMC40 Whitefly Whit fl SUSCEPTIBLE
    • A. socialis life cycle Time-course tissue Adult collection Eggs A. socialis stage Collection times after Pools infestation Adult Ad lt (1) 5 ho rs hours, 1 Egg (2) 7 days Nymph I instar (3) 14 days 2 Nymph II i t N h instar (4) 18 d days Nymph III instar (5) 20 days 3 Nymph IV instar (6) 27 days
    • Methodology Two technical replicates Three biological replicates Hybridization in two arrays: y y *Cassava UniGen set cDNA microarray ~ 5.300 clones *Cassava cDNA microarray for whitefly ~ 5 700 clones 5.700 72 hybridizations
    • Analysis Scanned using a ChipReader (BIO-RAD) Grids construction, spots identification VersArray analyzer (BIO-RAD) Normalization, standard regularization, elimination of outliers, N li ti t d d l i ti li i ti f tli replicate and dye swap analysis (MIDAS) Identification of differential expressed genes (SAM) Cluster Analysis (MeV) Bioinformatic analysis Cassava Unigene Microarray: 550 genes as significantly regulated in the six collect times and the two comparisons (resistant infested vs resistant non-infested & resistant infested vs susceptible infested), hi h i f t d) which 310 Up-regulated and 240 down-regulated. U l t d d d l t d Sequence annotation was done using GOMP. These ESTs sequences were compared to known protein sequences (The Arabidopsis Information Resource, TAIR) and mapped to Gene Ontology (GO) tterms and KEGG pathways using BLASTX Functional categories were defined using the d th i BLASTX. F ti l t i d fi d i th GO classification scheme.
    • Cluster analysis using MeV Hierarchical tree showing the differential genes in resistant genotype (MEcu-72) infested vs. non-infested, collects to 20 & 27 days post-infestation (Nymph III & IV). Cassava unigen set microarray Up regulated genes (207) Down regulated genes (80)
    • 7% 8% 14% 10% 1% Functional categories of regulated genes during cassava-whitefly interaction. Hybridizations hi fl i i b idi i were done with the Cassava Unigene Microarray
    • Specificity of defense responses Arabidopsis thaliana Frankliniella occidentalis (perforador-chupador) Spodoptera littoralis (generalista, masticador) Pieris rapae (especialista, masticador) Moscas blancas y áfidos (chupadores floemófagos)
    • Defense response to phloem-feeding insect (Order Hemiptera) SA (Salicylic Acid) JA/ET (Jasmonic Acid /Ethylene) Defense response
    • Ph N ot IP or 1: es 2 CY pi w -6 -5 -4 -3 -2 -1 at P7 0 1 2 3 4 5 ra er 14 tio n ch A1 an ox (N id AD ne or H l ed uc de N hy AD ta s e, dr og PH 2O en G At as H -F y2 e) yp e( ot II he ) tic ox al yg FA (S enD D pr aR -d ot se) ep ei en n de (t hi C ox nt orYP ed8 id Et ox 2 ox C or id ed hy or in2 uc lb ed ) ta en uc se ze ta , ne se zi de P nc -b hy R in dr X l di og ike ng e n -2 de as hy e dr og hibridization (Cassava Unigene Microarray) e Mn FaPse -a hy o po xid th or et ed al ic do al uct LP pr as /k D et 1 o t e, ei zi TR o n nc X- re SK D -b lik du S U e ct 1 F1 in Fe as 79 din rr e Th g d ed io eh ox re yd in do r 2- xi o g ox n en og Ah EI as lu pC N2 e ta -T ra SA te -d ep en de nt FA N D di D -d ox CY A1 ep yg P8 en en 6A de as1 nt e Oxidation_Reduction/Oxidative ox Be id C ta or Y -c edP7 m ar u c 6C ito ot ta 4 ch en P se on e er dr hy ox ia dr ire Oxidation Reduction/Oxidative Stress lp ox do ro yl xi ce as n ss e in 2 g AT pe G pt ST id U as 19 e be S ta KS su 6 ET bu FQ ni Ot U nk no w CR n C Pr D1 CH ot ei Su (C n pe O r ox PP ER AD id e CHATG C2 di AP ST sm ER U1 ut O 9 as N e E) [M n] AL , D m H ito 2B ch 7 on SA G dr 2a i1 l R CD 1 Genes differentially expressed in cassava in response to white fly attack through all the nymphal stages, determined by Microarray
    • tr an sc rip D t io N n A in bi iti C nd atal in iom g n od pr fa u ot ctlin ei or b n -8 -6 -4 -2 0 2 4 6 8 C2 -rin el d i atn do m egd ai n- co AT en nt AR do (C ai F nu ni 5H ng A1 cl C2 E ea pr se zi Ht oD /e nc ei xoa A3 n fin nrum ge clad r) eail o sle / Ri b /p e ci TA nF hoat u4s -c sp a t ha e n ta i sen G E RA A T IF 3 S M E fa SC PK m A g ily RE 13 tr CR an O IA sc W A7 rip -L tio IKAH E K en n 12 d on fa 3 , ct u cl or ea se /e xo nu nu EI cl cl N N 2 Signal transduction eo ea tid CR se F- /p YB yl K tr (C ho 3 an D sp sf PK ha G er -r At tas L3 as el e at RL e f a ed P7 m k ily in sc CK pr ase a r I1 ot ) ec ( ei r C n pr ow AS ot tr EIN ei an n sc KI ki de na rip NA ve se tio SR E lo fa n EIV pm m fa ) ily cto en pr r ta ot lly CRK ei re (C n gu D zi la PK nc te -r d el fin G at ge TP ed r pr -b ki ot in J na ei di n ng Ase8 Z ki pr ) na se ot ei Genes differentially expressed in cassava in response to white fly attack through all the fa n m A nymphal stages, determined by Microarray hibridization (Cassava Unigene Microarray) ily RR pr 9 ot ei n
    • Genes differentially expressed in cassava in response to white fly attack through all the nymphal stages, determined by Microarray hibridization (Cassava Unigene Microarray) GENE ID Putative function/homology Score(d) q-value(%) Defense Response Hypot. Protein Response to nematodes 1.21 0 Thaumatin SA Pathogenesis_related, Response to other organism 1.21 0 Lipase Defense Response to bacterium 1.2 0 PER21 peroxidase 21, Defense Response to fungus 1.1 0 RPN12A Peptidase, Response to auxin, cytoquinin, Proteasome 1 0 disease resistance protein (NBS-LRR cladefense response, Apoptosis 1 0 MTHSC70-2 (MITOCHONDRIAL HSP70 2), Response to virus, response to stress 1 0 CEV1 JA (CONSTITUTIVE EXPRESSION OF VSP 1);, Defense response 1 0 ATP-dependent Clp ATP d d t Cl protease, Defense response t D f 1 0 NPR3 SA (NPR1-LIKE PROTEIN 3);, Defense response to bacterium, fungus, incompatible interaction, response to SA 1 0 Esterase Defense response 1 0 CB5-D (CYTOCHROME B5 ISOFORM D); Defense response 1.9 7.5 ATNSI NUCLEAR SHUTTLE INTERACTING); N-acetyltransferase (interaction host-virus), Interspecies interaction 1.6 7.5 PAP3 (PURPLE ACID PHOSPHATASE 3); acid phosphatase/ protein serine/threonine phosphatase, Defense response 1.5 9.5 SCARECROW-LSA Response to chitin 2.4 3.3 EIN2 (ETHYLENE INSENSITIVE 2) t 2); transporter, C t l f t i signaling pathways regulated b ethylene (ET) d f t Central factor in i li th l t d by th l (ET), defense response 3.7 37 1.3 13 ATHCHIB (PR3) SA (ARABIDOPSIS THALIANA BASIC CHITINASE); chitinase (PR3), Killing of cell of another organism 5.8 6.8 ATPME3 pectinesterase, Response to nematodes 2.5 8 HSP81-2 (HEAT SHOCK PROTEIN 81-2), Defense response 3.8 1 acid phosphatase class B Defense response 3.6 1 AtRLP7 (Receptor Like Protein 7), Defense response 2.5 3.7 ATOSM34 SA (osmotin 34) (Thaumatin family), Defense response 2.1 4.4 Glucan endo 1 3 beta glucosidase 11 Defense response endo-1,3-beta-glucosidase 2.1 21 4.4 44 AFB2 (AUXIN SIGNALING F-BOX 2); auxin binding / ubiquitin-protein ligase, Defense response, response to auxin 2 5 ACD11 SA (ACCELERATED CELL DEATH 11); sphingosine transmembrane transporter, Defense response to baterium, response to SA 2 5 subtilase Serine protease -1 0.8 PSBO1 (PS II OXYGEN-EVOLVING COMPLEX 1); oxygen evolving/ poly(U) binding, Defense response, photoinhibition -2 7 disease resistance protein (NBS-LRR claDefense response -1.7 10 WHY3 (WHIRLY 3); DNA binding Defense response -1.6 10 CA1 (CARBONIC ANHYDRASE 1); carbonate dehydratase/ zinc ion binding, Defense response to bacterium binding -1.6 16 10 AFB2 (AUXIN SIGNALING F-BOX 2); auxin binding / ubiquitin-protein ligase, Defense response, response to auxin -1.3 10 VTC2 JA (vitamin c defective 2); GDP-D-glucose phosphorylase/ GDP-galactose:glucose-1-phosphate guanyltransferase, Defense respo -2.6 0 MLO10 (MILDEW RESISTANCE LOCUS O 10); calmodulin binding, Defense response, cell death -3.2 0 PLDBETA1 (PHOSPHOLIPASE D BETA 1); phospholipase D, Defense response to bacterium, incompatible interaction -3 3 NLA SA (nitrogen limitation adaptation); ubiquitin-protein ligase, Defense response to bacterium, response to SA, SAR -2.3 8 WHY3 (WHIRLY 3); DNA binding, Defense response -3.2 0 disease resistance protein (NBS LRR cla (NBS-LRR Defense response -2.8 28 1 JAZ8 JA (JASMONATE-ZIM-DOMAIN PROTEIN 8), Defense response, response to chitin , response to JA -2.5 1.5 SDF2 (STROMAL CELL-DERIVED FACTOR 2-LIKE PROTEIN PRECURSOR), Defense response to bacterium, fungus -2.3 1.7 PSBO1 (PS II OXYGEN-EVOLVING COMPLEX 1); oxygen evolving/ poly(U) binding, Defense response, photoinhibition -2.1 3.3 RCD1 JA (RADICAL-INDUCED CELL DEATH1); protein binding, Defense response to bacterium, response to JA, ET, superoxide -2 3.9
    • H yp ot . -4 -3 3 -2 -1 0 1 2 3 4 5 6 7 Pr te o di Th se in as au e m re at si in st Li an pa ce se pr ot PE ei R2 n (N RP 1 BS N -L 1 RR 2A cl M as TH s SC ) AT 70 -2 P- de CE pe nd V1 en t Cl p N PR Es 3 te ra se CB 5- D SC AT AR N hibridization (Cassava Unigene Microarray) EC SI RO PA W P3 -L IK E 13 AT , H CH EIN IB 2 (P R3 ac AT ) id ph PM E3 os ph H G SP lu at as 81 ca -2 n e en cl as do s -1 B ,3 At -b RL et P7 a- AT gl O uc SM os 34 id as di e se 11 as e AF re B2 Defense response si st AC an ce D 11 W pr su H ot bt Y3 ei ila (W n se H (N IR BS PS LY -L BO 3) RR 1 ; cl D as N A s) bi nd in g CA 1 AF B2 VT di C2 se as M e LO re PL 10 si D st BE an ce TA pr 1 ot ei N n LA (N BS W -L H RR Y3 cl as s) JA Z8 SD F2 PS BO 1 Genes differentially expressed in cassava in response to white fly attack through all the nymphal stages, determined by Microarray
    • GENE ID Putative function/homology Score(d) q-value(%) Cell Wall modification/organization/biosynthesis CEV1 (CONSTITUTIVE EXPRESSION OF VSP 1);Cellulose biosynthetic process, cell wall organization 1 0 ATPME1 pectinesterase. Cell wall modification, cell wall organization 2.8 6 ATPME3 pectinesterase. Cell wall modification, cell wall organization 2.5 6 Glucan endo-1,3-beta-glucosidase 11 Cellular cell wall organization 2.1 4.4 cinnamyl-alcohol dehydrogenase, cinnamyl alcohol dehydrogenase putati Phenylpropanoids biosynthesis -2.8 28 6 pectinacetylesterase, putative cellulose and pectin-containing cell wall -4.8 4 chitinase Cell wall catabolic process -2.3 6 chitinase Cell wall catabolic process -2.46 6 GENE ID Putative function/homology Score(d) q-value(%) Secondary Metabolism LUP1 Pentacyclic triterpenoid biosynthetic process 1 0 COP1 (CONSTITUTIVE PHOTOMORPHOGENIC 1); protein binding / ubiquitin-protein ligase, Flavonoid biosynthetic process 4 6 caffeoyl-CoA 3-O-methyltransferase y y Phenylpropanoid biosynthesis yp p y 1.4 6 CYP86A1 (CYTOCHROME P450 86 A1); fatty acid (omega-1)-hydroxylase/ oxygen binding, Phenylpropanoid biosynthesis 2.2 4.4 fumarate hydratase Catalitic activity -1 0.8 CAD cinnamyl-alcohol dehydrogenase, Phenylpropanoid biosynthesis -1.3 8 chitinase cell wall macromolecule catabolic process -2.3 8 caffeoyl CoA 3-O-methyltransferase Reinforcement of the plant cell wall under conditions that trigger the disease resistance response
    • Phenylpropanoids metabolic pathway showing four genes differentially expressed during the interaction- cassava whitefly
    • On-going work and Perspectives P i . Sequencing and bioinformatic analysis of experiments on Cassava whitefly library microarray. . Validation of candidates genes using real time PCR . Functional analysis of validate genes . Generate an integrated cassava metabolic model that explains the response to whitefly attack. . Cytological examination of the Hypersensitive response of whitefly- infested leaves to determine the specific location of callosa deposition and hypersensitive response to whitefly, ROS whitefly accumulation and Callose deposition in whitefly-infested leaves.
    • Acknowledgments . We acknowledge Gines-Mera fellowship for support the Ph D t di th Ph.D studies . Joe Tohme and Alfredo Alves . Biotechnology team