Genes differentially expressed in cassava in response to whitefly attack were identified through microarray analysis. Several genes involved in defense responses were found to be upregulated, including genes related to pathogenesis, proteasomes, chitinases, peroxidases, lipases, and heat shock proteins. Pathway analysis indicated that jasmonic acid/ethylene and salicylic acid signaling pathways regulating various defense mechanisms were activated. The microarray analysis provides insight into cassava's gene expression responses when defending against whitefly attack.

1. 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

2. General Objective
Understand the defense mechanisms of
cassava (Manihot esculenta Crantz) at the
gene expression level, in response to
whitefly (Aleurotrachelus socialis Bondar)
attack.

3. African Cassava Mosaic Disease
CMD

4. Neotropics
Whitefly:
y
Aleurotrachelus
socialis
Yield losses 5-
79%

5. Susceptible genotype
Source: Cassava Entomology, CIAT

6. MEcu-72 Whitefly RESISTANT
CMC40 Whitefly
Whit fl SUSCEPTIBLE

7. 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

8. 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

9. 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.

10. 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)

11. 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

12. Specificity of defense responses
Arabidopsis thaliana
Frankliniella occidentalis
(perforador-chupador) Spodoptera littoralis
(generalista, masticador)
Pieris rapae
(especialista,
masticador)
Moscas blancas y áfidos
(chupadores
floemófagos)

13. Defense response to phloem-feeding insect (Order
Hemiptera)
SA (Salicylic Acid)
JA/ET (Jasmonic Acid
/Ethylene)
Defense response

14. Ph N
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Genes differentially expressed in cassava in response to white fly attack through all the nymphal stages, determined by Microarray

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Genes differentially expressed in cassava in response to white fly attack through all the
fa n
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16. 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

17. H
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Genes differentially expressed in cassava in response to white fly attack through all the nymphal stages, determined by Microarray

18. 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

19. Phenylpropanoids metabolic pathway showing four
genes differentially expressed during the interaction-
cassava whitefly

20. 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.

21. Acknowledgments
. We acknowledge Gines-Mera fellowship for support
the Ph D t di
th Ph.D studies
. Joe Tohme and Alfredo Alves
. Biotechnology team