Isolation of genes differentially expressed during the defense response of Cassava
(Manihot esculenta Crantz) to whitefly (Aleurotrachelus socialis Bondar) attack
Bohorquez, Adriana1, Bernal, Diana1, Arias, Bernardo1, Bellotti, Anthony C.1 and Joe Tohme,1.
1CIAT, Agrobiodiversity and Biotechnology Project, AA6713, Cali, Colombia
INTRODUCTION METODOLOGY
Whiteflies can cause major yield losses in cassava in the Americas, Africa and Asia. Of the
different whiteflies species Aleurotrachelus socialis is the most damaging whitefly species in
northern South America. Systematic evaluation of the cassava germplasm bank (over 5,000
accessions) at CIAT resulted in the identification of cultivars with high levels of resistance to A.
socialis. A major source of host resistance in cassava is genotype M Ecu-72. In this work,
functional genomic tools (differential subtraction and microarrays) are used to investigate the
defense mechanisms of cassava in response to A. socialis attack, at the gene expression level. M
Ecu-72 (resistant) and CMC40 (susceptible) genotypes were infested with A. socialis adults using
clip cages, for the isolation of differentially expressed sequences using two strategies: First, the
genotype M Ecu-72, infested with A. socialis, was used as tester, while infested CMC40 was used
as driver to identify constitutively expressed resistance genes. Second, infested M Ecu-72 was
used as tester, while the driver was the same non-infested genotype to obtain genes implicated in
the downstream signaling cascade leading to the resistance phenotype. So far, a total of 6 different
subtractive cDNA libraries have been generated from leaves. Unique cDNA bands enriched or Bioessays (three Tissue collection (5 hours,
RNA isolation
cDNA synthesis Microarray Hybridization
mainly expressed in the resistant M Ecu-72 line have been isolated and cloned. These cDNAs biological replicate) 7, 14, 18, 20 and 27 days
cDNA Subtractive
might represent genes related with the resistance to A. socialis. Microarray-expression profiling post-infestation
Libraries
was used to identify putative, early response, regulatory, defense-related, and/or signaling genes
involved in the resistance of MEcu-72 against A. socialis.
RESULTS
Classification of white fly-regulated genes
In this study, changes in the Cassava transcriptome profile were examined throughout the life cycle of the whitefly, as changes in the plant defense gene RNAs occur in crop plants in response to
adult and nymphal stages. Three biological replicate experiments containing six plants per treatment were performed and the RNAs were pooled and hybridized to two replicate to Cassava
Unigene Microarray (CUM, 5376 unigene) (Lopez et al, 2004) and two replicate to Cassava Whitefly Microarray (CWfM, 5376 cDNA clones of subtractive libraries). To identify genes that were
significantly by white fly, the data were preprocessed using VersArray Analyzer® and MIDAS® for background adjustment and normalization. Hierarchical clustering was constructed using
MeV® (Figure 2). Significant Analysis of Microarray (SAM) software was used for differential analysis. SAM program identified in CUM 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), which 310 Up-regulated and 240 down-regulated. CUM sequence annotation
was done using GOMP (Rodriguez et al, unpublished). These ESTs sequences were compared to known protein sequences (The Arabidopsis Information Resource, TAIR) and mapped to Gene
Ontology (GO) terms and KEGG pathways using BLASTX. Functional categories were defined using the GO classification scheme (Figure 3). Twenty-one percent were of unknown function, no
match or “expressed proteins”. GO identified genes involved in Defense response (data shown below) , oxidative stress, transport, response to stimulus, proteolysis, cell wall modification,
photosynthesis, carbohydrate metabolism, etc. In secondary metabolism four genes belonging to the Phenylpropanoid Pathway were regulated (Figure 4). This pathway is related to reinforcement
of the plant cell wall under conditions that trigger the disease resistance response.
Genes involved in defense Up-regulated and Down-regulated (Underlined down-regulated)
Early responses (Egg, Nymph 1 and 2) Late responses (Nymph 3 and 4)
•Pathogenesis-related Thaumatin •CB5-D (CYTOCHROME B5 ISOFORM D)
•peroxidase 21 (PER21) (P21) (PRXR5) •ATNSI (NUCLEAR SHUTTLE INTERACTING); N-acetyltransferase (interaction host-virus)
•RPN12A Peptidase •PAP3 (PURPLE ACID PHOSPHATASE 3); acid phosphatase/ protein serine/threonine
phosphatase
•disease resistance protein (NBS-LRR class)
•HSP81-2 (HEAT SHOCK PROTEIN 81-2); ATP binding
•MTHSC70-2 (MITOCHONDRIAL HSP70 2)
•acid phosphatase class B family protein
•CEV1 (CONSTITUTIVE EXPRESSION OF VSP 1)
•AtRLP7 (Receptor Like Protein 7); kinase/ protein binding
•ATP-dependent Clp protease
•ATOSM34 (osmotin 34) (Thaumatin family)
•NPR3 (NPR1-LIKE PROTEIN 3)
•Glucan endo-1,3-beta-glucosidase 11
•Esterase
•AFB2 (AUXIN SIGNALING F-BOX 2); auxin binding / ubiquitin-protein ligase
•SCARECROW-LIKE 13,
•ACD11 (ACCELERATED CELL DEATH 11); sphingosine transmembrane transporter
•EIN2 (ETHYLENE INSENSITIVE 2); transporter
•PSBO1 (PS II OXYGEN-EVOLVING COMPLEX 1); oxygen evolving/ poly(U)
•ATHCHIB (ARABIDOPSIS THALIANA BASIC CHITINASE); chitinase (PR3)
• disease resistance protein (NBS-LRR class), putative
•ATPME3; pectinesterase
•WHY3 (WHIRLY 3); DNA binding
•PAD1 (20s proteasome alpha subunit pad1); endopeptidase/ peptidase/ threonine-type
endopeptidase •AFB2 (AUXIN SIGNALING F-BOX 2); auxin binding / ubiquitin-protein ligase
•subtilase family protein •VTC2 (vitamin c defective 2);
•MLO10 (MILDEW RESISTANCE LOCUS O 10); calmodulin binding •JAZ8 (JASMONATE-ZIM-DOMAIN PROTEIN 8)
•PLDBETA1 (PHOSPHOLIPASE D BETA 1); phospholipase D •SDF2 (STROMAL CELL-DERIVED FACTOR 2-LIKE PROTEIN PRECURSOR)
•NLA (nitrogen limitation adaptation); ubiquitin-protein ligase •RCD1 (RADICAL-INDUCED CELL DEATH1)
Phenylpropanoids biosynthesis: Phenylpropanoids are a group of plant secondary metabolites derived from phenylalanine and having a wide Figure 2: Hierarchical tree derivative of SAM showing some of the
variety of functions both as structural and signaling molecules. Phenylalanine is first converted to cinnamic acid by deamination. It is followed by genes s significantly regulated during cassava-whitefly interaction at
hydroxylation and frequent methylation to generate coumaric acid and other acids with a phenylpropane (C6-C3) unit. Reduction of the CoA- nymph 3 and nymph 4 stages between three biological replicate of
activated carboxyl groups of these acids results in the corresponding aldehydes and alcohols. The alcohols are called monolignols, the starting MEcu-72 infested (I-R) Vs. infested CMC40 (I-S) genotypes.
compounds for biosynthesis of lignin.
Figure 4:
Phenylpropanoids
metabolic pathway
showing four genes Figure 3: Functional categories of regulated
differentially expressed genes during cassava-whitefly interaction.
during the interaction- Hybridizations were done with the Cassava
cassava whitefly Unigene Microarray (CUM, 5376 unigenes)
ONGOING WORK ACKNOWLEDGMENT
-Sequences analysis of genes regulated obtained from Cassava Whitefly Microarray.
-We acknowledge Gines-Mera fellowship and Fausto Rodriguez for his technical
-Real-Time PCR validation of candidate genes. support.