2. Biotech - IJBTR -Identification of differentially expressed genes - Inas Farouk Fahmy.pdf

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International Journal of Bio-Technology

and Research (IJBTR)

ISSN(P): 2249-6858; ISSN(E): 2249-796X

Vol. 5, Issue 3, Jun 2015, 9-26

© TJPRC Pvt. Ltd.

IDENTIFICATION OF DIFFERENTIALLY EXPRESSED GENES IN VIRULIFEROUS

BEMI SIA TABACI USING DIFFERENTIAL DISPLAY ACP-RT-PCR

INAS F. FAHMY1, ASMAA F. DARWEESH

2, MOHMED A. EL-SATAR

3, RANIA M. ABOU-ALI

4, AHMED

H. M. ELWAHY5

1,2,Department of Microbial molecular biology, Agriculture Genetic Engineering Research Institute, Giza, Egypt

3Department of Molecular Biology, Agriculture Genetic Engineering Research Institute, Giza, Egypt

4Department of Nucleic acid Structure and Function, Agriculture Genetic Engineering Research Institute, Giza, Egypt

5Department of chemistry, Faculty of Science, Cairo University, Giza, Egypt

ABSTRACT

Acquisition of plant viruses has different effects on physiological mechanisms in vector insects and its

endosymbionts. Bemisia tabaci is the only known vector of Tomato yellow leaf curl virus (TYLCV), which is considered

to be the most serious virus affecting tomato crop in tropical and subtropical regions. In this study we aimed at studying the

expression profile of the non-viruliferous and viruliferous whiteflies reared on TYLCV. We used the annealing control

primer (ACP)-differential display RT-PCR method to identify differentially expressed fragments (DEFs) that may

contribute in virus translocation, thus transmission of virus via its vector whitefly. Using four arbitrary ACP primers, a

total of 14 DEFs were identified and sequenced. BLAST searches revealed high homology with some putatively

transmission related genes like endosymbionts 16S ribosomal RNA genes, (StAR)-related lipid transfer (START),

peptidase and Vitellogenin. Some other fragments have showed a considerable homology with a former studied EST

fragments registered in the Genbank. The data obtained were confirmed using real time-PCR analysis and revealed that the

expression of all endosymbionts 16S ribosomal RNA gene in case of the viruliferous whiteflies showed a significant down-

regulation, while the steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) protein, peptidase and

Vitellogenin genes showed up-regulation in case of viruliferous whiteflies.

KEYWORDS: ACP DD-RT-PCR, Differential Expressed fragments (DEFs), TYLCV, whitefly and endosymbionts

bacteria

INTRODUCTION

The whitefly Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a cryptic species complex composed of at

least 24 morphologically differentiated species (Hu et al. 2011; De Barro et al. 2011; Shadmany et al. 2013; Luan et al.

2011). Bemisia tabaci causes a huge damage by direct feeding on plants and indirect by being a vector of more than 100

different species of begomoviruses (Alemandri et al. 2012).The relationship between begomoviruses and whiteflies is very

complicated. For example, Tomato yellow leaf curl virus (TYLCV) is a geminivirus belongs to the genus Begomovirus

from Geminiviridae family. TYLCV is the causal agent of Tomato Yellow Leaf Curl Disease (TYLCD), which is the most

devastating viral disease affecting tomato worldwide including Egypt and all countries of the Middle East and the

Mediterranean basin. B. tabaci can transmit 115 species of begomoviruses in a persistent-circulative manner (Hogenhout et

al. 2008). Begomoviruses and whiteflies have been used as good model to study plant, virus and insect vector interactions

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Identification of Differentially Expressed Genes in Bemisiatabaci using differential Display ACP-RT-PCR 11


Cloning and Sequence Analysis

Differentially expressed bands were extracted and cloned into pGEM-T easy vector (Promega, USA) transformed

into DH5α ( Escherichia coli) competent cells (Invitrogen). To confirm the identities of insert DNA, isolated plasmids were

sequenced by one direction T7 forward primer (National Instrumentation Center for Environmental Management, Korea)

using an ABI3730XL sequencer (Applied Biosystems).Sequences were analyzed by searching for similarities using a

BLASTx and BLASTn programs(Altschul et al. 1990) at the National Center for Biotechnology Information (NCBI).

Quantification of the Targeted Genes by Real Time-PCR

The qRT-PCR was performed in a AB7300 Real-Time PCR System® (Applied Biosystems, USA) using SYBR®

Premix ExTaqII™ from TAKARA (Catalog no. R R82SW, TAKARA, Japan). Specific primers to amplify the targeted

gene were designed using DNA integrated technologies online software for qPCR primer design tool. The fragments were

amplified by using the primer pairs see (Table 3) in both viruliferous and non-viruliferous whitefly in order to compare the

expression of the viruliferous genes. The HSP90 house keeping gene was used as an endogenous control(Li et al. 2013).

The thermal cycler program was: first step (as a hot start step) at 95ºC for 30 seconds, followed by second step which

consisting from 43 cycles of 95ºC for 5 seconds, 60ºC for 34 seconds, then the melting curve step of 95ºC for 15 seconds,

60ºC for 1 minute and 95ºC for 15 seconds.

RESULTS

Identification of DEGs in viruliferous and Non-viruliferous whitefly

A differential display analysis was performed employing ACP dd RT-PCR to isolate any DEGs in viruliferous

and non-viruliferous whitefly using a combination of 4 arbitrary primers and one anchored oligo (dT) primers. A total of

14 DEG fragments were isolated from gels and sequenced. Out of the 14 identified fragments, six were up- regulatedand

eight other fragmentswere down-regulated in the viruliferous insect compared with the non-viruliferous one (Table 2).

Sequence Analysis of the differentially Displayed Gene Fragments

BLASTN and BLASTX searches of all 14 sequences against the GenBank database revealed that8 of these DEGs

have been well characterized in other species; the remaining fragments did not have significant sequence homology with

those of existing genes in GenBank. Clone ACP14-1 whose expression was down- regulated, showed 83% significant

homology with Whitefly Bemisia tabaci (reared on TYLCV infected plants) cDNA library Bemisia tabaci accession

number EE602294.1 with E value of 0.0; clone ACP14-2 whose expression was down- regulated, showed 99% significant

homology with Primary endosymbiont of Bemisia tabaci clone KEN3 16S ribosomal RNA gene, partial sequence of

accession number AF400460.1 with E value of 1e-67

; clone ACP14-4 whose expression was down- regulated, showed 99%

significant homology with Candidatus Portiera aleyrodidarum BT-B-HRs strain BT-B 16S ribosomal RNA of accession

number NR_102830.1 with E value of 0.0; ACP14-7 was found to show partial homology with Vitellogenin clone Q of

B.tabaci registered in the gene bank under accession number GU332722.1 with E value of 5e-34

and 81% identity. clone

ACP20-2 whose expression was up- regulated, showed 32% significant homology with stAR-related lipid transfer protein

7, mitochondrial [ Acyrthosiphon pisum] of accession number XP_001943914.1 with E value of 0.043 because the gene is

very long so the similarity percentage was low due to the small fragment discovered through the blast. ACP22-1 has a

significant homology with clone BT-TYLCV-060-1-H1-T3_H01 Whitefly Bemisia tabaci (reared on TYLCV infected

plants) cDNA library Bemisia tabaci cDNA 5', mRNA sequence accession no EE602081.1 and EE600846.194% with E

http://www.tjprc.org/http://www.tjprc.org/http://www.ncbi.nlm.nih.gov/nucleotide/113017961?report=genbank&log$=nucltop&blast_rank=1&RID=EVKP2JE501Rhttp://www.ncbi.nlm.nih.gov/nucleotide/18029912?report=genbank&log$=nucltop&blast_rank=1&RID=XN8T4CVZ01Rhttp://www.ncbi.nlm.nih.gov/nucleotide/507148023?report=genbank&log$=nucltop&blast_rank=1&RID=XNKCV8KR014http://www.ncbi.nlm.nih.gov/nucleotide/315307981?report=genbank&log$=nucltop&blast_rank=1&RID=HJV7J11W013http://www.ncbi.nlm.nih.gov/protein/193615447?report=genbank&log$=prottop&blast_rank=2&RID=FVJ213G0015http://blast.ncbi.nlm.nih.gov/blast/Blast.cgi#alnHdr_113020438http://blast.ncbi.nlm.nih.gov/blast/Blast.cgi#alnHdr_113020438http://blast.ncbi.nlm.nih.gov/blast/Blast.cgi#alnHdr_113020438http://blast.ncbi.nlm.nih.gov/blast/Blast.cgi#alnHdr_113020438http://www.ncbi.nlm.nih.gov/nucleotide/113020438?report=genbank&log$=nucltop&blast_rank=1&RID=J5JND6MK01Rhttp://www.ncbi.nlm.nih.gov/nucleotide/113019186?report=genbank&log$=nucltop&blast_rank=2&RID=J5JND6MK01Rhttp://www.ncbi.nlm.nih.gov/nucleotide/113019186?report=genbank&log$=nucltop&blast_rank=2&RID=J5JND6MK01Rhttp://www.ncbi.nlm.nih.gov/nucleotide/113020438?report=genbank&log$=nucltop&blast_rank=1&RID=J5JND6MK01Rhttp://blast.ncbi.nlm.nih.gov/blast/Blast.cgi#alnHdr_113020438http://blast.ncbi.nlm.nih.gov/blast/Blast.cgi#alnHdr_113020438http://www.ncbi.nlm.nih.gov/protein/193615447?report=genbank&log$=prottop&blast_rank=2&RID=FVJ213G0015http://www.ncbi.nlm.nih.gov/nucleotide/315307981?report=genbank&log$=nucltop&blast_rank=1&RID=HJV7J11W013http://www.ncbi.nlm.nih.gov/nucleotide/507148023?report=genbank&log$=nucltop&blast_rank=1&RID=XNKCV8KR014http://www.ncbi.nlm.nih.gov/nucleotide/18029912?report=genbank&log$=nucltop&blast_rank=1&RID=XN8T4CVZ01Rhttp://www.ncbi.nlm.nih.gov/nucleotide/113017961?report=genbank&log$=nucltop&blast_rank=1&RID=EVKP2JE501Rhttp://www.tjprc.org/


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12 Inasf. Fahmy, Asmaa F. Darweesh, Mohmed A. El-Satar, Rania M. Abou-Ali, Ahmed H. M. Elwahy

Impact Factor (JCC): 3.1245 NAAS Rating: 2.75

value of 2e-158

and 85% query coverage. ACP22-2 whose expression was up- regulated, showed 83% significant homology

with peptidase S14 [ Plautia stali symbiont] under accession number WP_041387620with E value of 3e-19.ACP32-2 has

revealed a significant homology with EST T_TYLCV001_A04 Whitefly Bemisia tabaci (reared on TYLCV infected

plants) cDNA library Bemisia tabaci cDNA 5', mRNA sequence of accession number EE599909.1with E value of 3e-66and 85% identity . Clones ACP14-3, ACP14-5, ACP14-6, ACP20-1, ACP32-1 and ACP-32-3 did not have significant

sequence homology with those of existing genes in GenBank (Table 2 and 4) & (Fig 2, 3, 4and 5).

Quantitative Real-time PCR Confirmation of Selected Genes

To confirm the results of the DGEs analyses, the expression of 5 selected genes was analyzed using qPCR. cDNA

was synthesized using SmartScribe cDNA synthesis kit Clontech. qPCR was done using the SYBR PrimeScript reverse

transcription-PCR (RT-PCR) kit II (TliRNaseH Plus) (Takara). qPCRs were carried out on the ABI Prism 7500 fast real-

time PCR sys2e-158tem (Applied Biosystems) using SYBR green based detection. Each gene was analyzed in triplicate,

after which the average threshold cycle (Ct) was calculated per sample. The relative expression levels were calculatedusing the 2

−ΔΔCT method. As an endogenous control, the expression of HSP90 was measured in parallel. In this study, qPCR

assay showed that the transcript of the HSP90 gene is at the same level in both the viruliferous and non-viruliferous

whiteflies, indicating that the expression of the HSP90 gene was unaffected by TYLCV. In RT-PCR the reaction is

detected by accumulating the fluorescent signal and the detection was measured by the threshold cycle (Ct) which is the

number of required cycles for the fluorescent signal to cross the threshold of the background level. Ct values are inversely

proportional to the amount of template in the sample. The results were consistent with the ACP ddRT-PCR prescreening

(Fig. 6, 7, 8 and 9) & (table5).The mRNA expressions of primary and candidatus endosymbionts 16S ribosomal RNA

genes were significantly higher at the non-viruliferous whiteflies compared to the viruliferous whiteflies. In contrast the

expressions of START protein, peptidase, vitellogenin were significantly higher in the viruliferous whiteflies.

DISCUSSIONS

This study was performed using the PCR based ddRT-PCR method; formerly the Gene Hunter first designed

primersproved to produce somehow a high rate of false positives(Liang & Pardee 1992). A new annealing control primer

(ACP) differential display PCR method was developed. Annealing control oligo-dT primers (dT-ACP1 and dT-ACP2)

were used for the synthesis of first-strand cDNA and subsequent PCR amplification to ensure accurate annealing. Using

the ACP ddRT-PCR method, eight fragments could be involved in transmission of TYLCV via whitefly were identified.

Six fragments belonged to B. tabaci endosymbiotic bacteria and two fragments belonged to whitefly insect itself (Table

4).TYLCV009_C12 Whitefly Bemisia tabaci (reared on TYLCV infected plants) cDNA library Bemisia tabaci;BT-

TYLCV-060-1-H1-T3_H01 Whitefly Bemisiatabaci (reared on TYLCV infected plants) cDNA library Bemisia tabacic;

EST T_TYLCV001_A04 Whitefly Bemisiatabaci (reared on TYLCV infected plants) cDNA library Bemisia tabacic DNA

Searching of this cDNA libraries showed that it is related to Candidatus endosymbiont of Bemisia tabaci whitefly

(Leshkowitz et al. 2006), but no function of this library has been yet identified as all blastx, tblastx and searching in

conserved domain showed no result for this library. It may be still under investigation. The fragments of the Primary

endosymbiont 16SrRNA bacterial symbionts in insects can influence many functions of their host including nutrition, host-

plant utilization, parasitoid resistance, reproductive manipulation and ability to cope with environmental factors such as

heat stress (Brownlie & Johnson 2009). Primary symbionts benefit host insects by aiding in digestion of food or by providing nutrients that are lacking in the diet of insect. Secondary symbionts, in contrast, may not be required for host

http://www.ncbi.nlm.nih.gov/nucleotide/113015217?report=genbank&log$=nucltop&blast_rank=1&RID=J57SPKTS016http://blast.ncbi.nlm.nih.gov/blast/Blast.cgi#alnHdr_113020438http://blast.ncbi.nlm.nih.gov/blast/Blast.cgi#alnHdr_113020438http://blast.ncbi.nlm.nih.gov/blast/Blast.cgi#alnHdr_113020438http://blast.ncbi.nlm.nih.gov/blast/Blast.cgi#alnHdr_113020438http://blast.ncbi.nlm.nih.gov/blast/Blast.cgi#alnHdr_113020438http://blast.ncbi.nlm.nih.gov/blast/Blast.cgi#alnHdr_113020438http://blast.ncbi.nlm.nih.gov/blast/Blast.cgi#alnHdr_113020438http://www.ncbi.nlm.nih.gov/nucleotide/113015217?report=genbank&log$=nucltop&blast_rank=1&RID=J57SPKTS016


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survival but may play important roles in host biology and evolution (Himler et al. 2011). Whiteflies harbor an obligatory

symbiotic bacterium – Candidatus Portiera aleyrodidarum and diverse facultative bacteria which may include

Hamiltonella, Arsenophonus, Fritchea, Wolbachia and Rickettsia (Everett et al. 2005) A lot of researches have speculated

that the S-symbiont. Hamiltonella was probably associated with the transmission efficiency of TYLCV by the whiteflyvector (Gottlieb et al. 2010). Israeli Q biotype lacks Hamiltonella and cannot effectively transmit TYLCV, while Chinese

Q frequently contains Hamiltonella and can efficiently transmit TYLCV to tomato (Gottlieb et al. 2010). The presence of

Hamiltonella is involved in acquisition, retention, and transmission of TYLCV by B. tabaci Q and in significant

differences for TYLCV accumulation in plants exposed to the whiteflies. On the other hand, virus transmission efficiency

seems to be more related with differences in symbiont composing than with whitefly biotypes, regardless several literature

stressing the importance of the biotypes in viral transmission (Su et al. 2013). Also whitefly symbionts may play important

roles in the biology of their hosts, including resistance to parasitoids and susceptibility to insecticides (Mahadav et al.

2008). Our expression level analysis of primary and candidates 16S ribosomal genes showed significant down- regulation

in case of viruliferous whitefly and this result is consistent with the study of gene expression profiling of the whitefly

( Bemisia tabaci ) Middle East – Asia Minor 1 feeding on healthy and Tomato yellow leaf curl China virus-infected

tobacco.They showed in their study that the expression level of Primary endosymbiont 16S ribosomal RNA gene was

significantly down regulated in case of viruliferous whitefly(Li et al. 2011).Furthermore, the involvement of symbioti c

bacteria in their insect host’s response to virus infection has been shown in Drosophila, in which Wolbachia increases D.

melanogaster resistance to Drosophila C virus and two other RNA virus infections (Nora virus and Flock House virus),

reducing the load of viruses in infected flies(Teixeira et al. 2008). These findings together with our observation strongly

support the hypothesis that endosymbionts are involved in the whitefly response to various environmental stresses,

including virus infection.

Vitellogenin is an egg yolk precursor protein expressed in the females of nearly all oviparous species is the

precursor of the lipoproteins and phospho-proteins that make up most of the protein content of yolk (Robinson 2008).

During vitellogenesis, Vg is produced mostly in female fat body, secreted into the hemolymph and accumulated in the

developing oocytes by a receptor-mediated way during oogenesis(Tufail& Takeda 2009). Vitellogenesis can be affected by

many factors, including hormone, stress and nutrition (Shu et al. 2009). It was demonstrated that MEAM1 benefits from

the indirect virus mutualism in some manner associated with ovarian development (Guo et al. 2010). This benefit was

assumed due to the enhanced synthesis and uptake of yolk protein. It was showed that levels of hemolymph Vg and ovary

Vg in the females fed on virus-infected tobacco plants were significantly higher than those in females fed on uninfected

tobacco plants(Guo et al. 2012). It was also demonstrated that the level of Vg gene is increased in whiteflies feeding on

TYLCCNV+TYLCCNB co-infected plants when compared with that of whiteflies feeding on either EHA105-infiltrated or

TYLCCNV-infected plants. In addition, it seemed that the indirect virus mutualism has effects only on the synthesis and

uptake of Vg but not the timing of vitellogenesis in whiteflies (Guo et al. 2010).These data together reveal that MEAM1

individuals fed on virus-infected plants improved the levels of Vg synthesis and uptake, and also they demonstrated the

similar Vg gene transcript levels between whiteflies feeding on artificial diets containing purified TYLCCNV, inactivated

TYLCCNV with only phosphate buffer. Our expression level analysis results showed that the Vg was significantly up-

regulated in case of viruliferous whitefly, this suggest that TYLCV uptake by whitefly insect lead to the enhancement of

the expression level of Vg, as virus translocation could be treated as one of the stress factors that a ffect Vg production.

http://www.tjprc.org/http://www.tjprc.org/http://en.wikipedia.org/wiki/Egg_yolkhttp://en.wikipedia.org/wiki/Oviparoushttp://en.wikipedia.org/wiki/Lipoproteinshttp://en.wikipedia.org/wiki/Phosphoproteinhttp://en.wikipedia.org/wiki/Phosphoproteinhttp://en.wikipedia.org/wiki/Lipoproteinshttp://en.wikipedia.org/wiki/Oviparoushttp://en.wikipedia.org/wiki/Egg_yolkhttp://www.tjprc.org/


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Peptidase is an enzyme that performs proteolysis, it begins proteincatabolism by hydrolysis of the peptide bonds

that link amino acids together in a polypeptide chain. Proteases have evolved multiple times, and different classes of

protease can perform the same reaction by completely different catalytic mechanisms. Proteases can be found in animals,

plants, bacteria, archaea and viruses. Various proteolytic enzymes/systems have been described that are able to disarmviruses by degrading their major proteins.Among them, the ubiquitin 26S proteasome degradationsystem, most genes

involved in translation, ribosome, spliceosome, aminoacyl-tRNA biosynthesis and amino acid metabolism were down-

regulated in the viruliferous whiteflies, indicating that the protein synthesis and amino acid metabolism of the viruliferous

whitefly were inhibited by TYLCCNV (Luan et al. 2011). So we can postulate that TYLCV affect the regulation of the

peptidase enzyme causing inhibition of the protein synthesis. Our findings is consistent with the result obtained formerly

by (Luan et al. 2011)of Transcriptional Response of Whitefly to Tomato Yellow Leaf Curl China Virus, they found that 4

genes belonged to ubiquitin-proteasome pathway were showed significant up – regulation in case of infected whitefly (Luan

et al. 2011).

Steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domains were first identified in

cholesterol-binding StAR orthologs from mammals. They are also found in plant proteins, where they are predicted to

mediate transport and signaling of lipidsSTART protein is a protein domain spanning =210 residues (Venkata & Schrick

2006). It is conserved protein in plants and animals and serves as a binding interface for lipids that function in many

different processes (Soccio & Breslow 2003; Schrick et al. 2004).The START domain plays as a shield to protect a

hydrophobic lipid from a hydrophilic environment. It acts as a lipid-exchange. START proteins are involved in different

biological processes as lipid metabolism, which involves START proteins that contain thioesterase catalytic activities; lipid

transfer between cellular compounds and signal transduction, which involves the RhoGAP START proteins (Alpy &

Tomasetto 2005). START protein involved in ecdysteroid biosynthesis is positively correlated with the rise in ecdysteroid production by ovaries of a female insect, as is a key gene in development process of the insect and eggs production

(Sieglaff et al. 2005). Our expression level of START domain showed significant up- regulation in case of viruliferous

whitefly, suggestion that TYLCV induce the expression of START domain. This induction play important role in

increasing eggs production. It was demonstrated that MEAM1 benefits from the indirect virus mutualism in some manner

associated with ovarian development (Guo et al. 2010). DD-RT-PCR using ACP primers sets could reveal some

mysterious genes induced during virus translocation after obtaining the full sequence of these fragments using RACE-

technique as has been done by in a former study by (Rahman et al. 2013), subsequently followed by silencing experiments

in order to discover the effective identified gene fragments which give the best effect on preventing and or eliminating

virus translocation and transmission by their whitefly vector.

CONCLUSIONS

TYLCV infection can significantly change the gene expression profile of the whitefly through direct and indirect

effects. The changes of gene expression pattern in whitefly and presence of TYLCV affect the fecundity and longevity of

whitefly feed on the virus-infected plants. Further analysis on the discovered fragments which showed no homology to

known sequences should be done by obtaining longer fragments which can be then homologues to other known sequences.

Also the fragments that showed homology with cDNA library of whitefly reared on TYLCV should be further analyzed by

obtaining longer fragments and studying their effect by gene knockdown strategies. Functional genomics studies could

then reveal the effect of these fragments on virus translocation and or transmission. Thus employing RNAi strategy using

http://en.wikipedia.org/wiki/Enzymehttp://en.wikipedia.org/wiki/Proteolysishttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Hydrolysishttp://en.wikipedia.org/wiki/Peptide_bondhttp://en.wikipedia.org/wiki/Amino_acidhttp://en.wikipedia.org/wiki/Polypeptidehttp://en.wikipedia.org/wiki/Convergent_evolutionhttp://en.wikipedia.org/wiki/Catalytic_mechanismhttp://en.wikipedia.org/wiki/Animalhttp://en.wikipedia.org/wiki/Planthttp://en.wikipedia.org/wiki/Bacteriahttp://en.wikipedia.org/wiki/Archaeahttp://en.wikipedia.org/wiki/Virushttp://en.wikipedia.org/wiki/Virushttp://en.wikipedia.org/wiki/Archaeahttp://en.wikipedia.org/wiki/Bacteriahttp://en.wikipedia.org/wiki/Planthttp://en.wikipedia.org/wiki/Animalhttp://en.wikipedia.org/wiki/Catalytic_mechanismhttp://en.wikipedia.org/wiki/Convergent_evolutionhttp://en.wikipedia.org/wiki/Polypeptidehttp://en.wikipedia.org/wiki/Amino_acidhttp://en.wikipedia.org/wiki/Peptide_bondhttp://en.wikipedia.org/wiki/Hydrolysishttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Proteinhttp://en.wikipedia.org/wiki/Proteolysishttp://en.wikipedia.org/wiki/Enzyme


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effective genes could reveal the effect of virus translocation and or blockage using the newly identified fragments through

this study.

ACKNOWLEDGEMENTS

We would like to extend our deep gratitude to the STDF (Science and Technology Development Fund), Egyptian

Academy of Science, grant no# 892 for financing this part of the project and for helping in publishing this part of the study,

and Dr. Khalid Dougdoug for his scientific support and the collection of TYLCV samples used in establishing the

infectious clone of the virus.

REFERENCES

1. Alemandri, V. et al., (2012). Species Within the Bemisia tabaci (Hemiptera: Aleyrodidae) Complex in Soybean

and Bean Crops in Argentina. Journal of Economic Entomology, 105(1), pp.48 – 53. Available at:

http://dx.doi.org/10.1603/EC11161 [Accessed March 19, 2015].

2. Alpy, F. & Tomasetto, C., (2005). Give lipids a START: the StAR-related lipid transfer (START) domain in

mammals. Journal of cell science, 118(Pt 13), pp.2791 – 801. Available at:

http://jcs.biologists.org/content/118/13/2791.full#ref-71 [Accessed March 17, 2015].

3. Altschul, S.F. et al., (1990). Basic local alignment search tool. Journal of molecular biology, 215(3), pp.403 – 10.

Available at: http://www.ncbi.nlm.nih.gov/pubmed/2231712 [Accessed July 10, 2014].

4. Anon, (2007). Tomato Yellow Leaf Curl Virus Disease: Management, Molecular Biology, Breeding for

Resistance, Springer Science & Business Media. Available at:

http://books.google.com/books?hl=en&lr=&id=8NR12W1-B0IC&pgis=1 [Accessed March 19, 2015].

5. De Barro, P.J. et al.,( 2011). Bemisia tabaci: a statement of species status. Annual review of entomology, 56, pp.1 –

19. Available at: http://www.ncbi.nlm.nih.gov/pubmed/20690829 [Accessed March 19, 2015].

6. Baumann, P., (2005). BIOLOGY OF BACTERIOCYTE-ASSOCIATED ENDOSYMBIONTS OF PLANT SAP-

SUCKING INSECTS. Annual Review of Microbiology, 59(1), pp.155 – 189. Available at:

http://www.ncbi.nlm.nih.gov/pubmed/16153167 [Accessed March 16, 2015].

7. Brownlie, J.C. & Johnson, K.N., (2009). Symbiont-mediated protection in insect hosts. Trends in microbiology,

17(8), pp.348 – 54. Available at: http://www.ncbi.nlm.nih.gov/pubmed/19660955 [Accessed January 28, 2015].

8. Czosnek, H. & Ghanim, M., (2002). The circulative pathway of begomoviruses in the whitefly vector Bemisia

tabaci - insights from studies with Tomato yellow leaf curl virus. Annals of Applied Biology, 140, pp.215 – 231.

Available at: ://WOS:000177249800001.

9. Everett, K.D.E. et al., (2005). Novel chlamydiae in whiteflies and scale insects: Endosymbionts “Candidatus

Fritschea bemisiae” strain Falk and “Candidatus Fritschea eriococci” strain Elm. International Journal of

Systematic and Evolutionary Microbiology, 55, pp.1581 – 1587.

10. Gottlieb, Y. et al., (2010). The transmission efficiency of tomato yellow leaf curl virus by the whitefly Bemisia

tabaci is correlated with the presence of a specific symbiotic bacterium species. Journal of virology, 84(18),

pp.9310 – 7. Available at:



8/18



http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2937599&tool=pmcentrez&rendertype=abstract

[Accessed March 16, 2015].

11. Guo, J.-Y. et al., (2010). An invasive whitefly feeding on a virus-infected plant increased its egg production and

realized fecundity. PloS one, 5(7), p.e11713. Available at:


[Accessed February 26, 2015].

12. Guo, J.-Y. et al., (2012). Enhanced vitellogenesis in a whitefly via feeding on a begomovirus-infected plant. PloS

one, 7(8), p.e43567. Available at:

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0043567#pone.0043567-Tufail1 [Accessed

February 26, 2015].

13. Hanssen, I.M., Lapidot, M. & Thomma, B.P.H.J., (2010). Emerging Viral Diseases of Tomato Crops. Molecular

Plant-Microbe Interactions, 23(5), pp.539 – 548. Available at:

http://apsjournals.apsnet.org/doi/abs/10.1094/MPMI-23-5-0539 [Accessed March 19, 2015].

14. Himler, A.G. et al., (2011). Rapid Spread of a Bacterial Symbiont in an Invasive Whitefly Is Driven by Fitness

Benefits and Female Bias. Science, 332(6026), pp.254 – 256. Available at:

http://www.ncbi.nlm.nih.gov/pubmed/21474763 [Accessed March 16, 2015].

15. Hogenhout, S.A. et al., (2008). Insect vector interactions with persistently transmitted viruses. Annual review of

phytopathology, 46, pp.327 – 59. Available at:

http://www.annualreviews.org/doi/abs/10.1146/annurev.phyto.022508.092135 [Accessed February 9, 2015].

16. Hu, J. et al., (2011). An Extensive Field Survey Combined with a Phylogenetic Analysis Reveals Rapid and

Widespread Invasion of Two Alien Whiteflies in China R. G. Roberts, ed. PLoS ONE , 6(1), p.e16061. Available

at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3025023&tool=pmcentrez&rendertype=abstract


17. Kao, R.H. et al., (2003). Application of differential display, with in situ hybridization verification, to microscopic

samples of breast cancer tissue. International journal of experimental pathology, 84(5), pp.207 – 12. Available at:



18. Leshkowitz, D. et al., (2006). Whitefly (Bemisia tabaci) genome project: analysis of sequenced clones from egg,

instar, and adult (viruliferous and non-viruliferous) cDNA libraries. BMC genomics, 7(1), p.79. Available at:

http://www.biomedcentral.com/1471-2164/7/79 [Accessed March 10, 2015].

19. Li, J.-M. et al., (2011). Gene expression profiling of the whitefly (Bemisia tabaci) Middle East - Asia Minor 1

feeding on healthy and Tomato yellow leaf curl China virus-infected tobacco. Insect Science, 18(1), pp.11 – 22.

Available at: http://doi.wiley.com/10.1111/j.1744-7917.2010.01386.x [Accessed February 19, 2015].

20. Li, R. et al., (2013). Reference gene selection for qRT-PCR analysis in the sweetpotato whitefly, Bemisia tabaci

(Hemiptera: Aleyrodidae). PloS one, 8(1), p.e53006. Available at:http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0053006 [Accessed April 9, 2015].


9/18



21. Liang, P., Averboukh, L. & Pardee, A.B., (1993). Distribution and cloning of eukaryotic mRNAs by means of

differential display: refinements and optimization. Nucleic acids research, 21(14), pp.3269 – 75. Available at:



22. Liang, P. & Pardee, A.B., (1992). Differential display of eukaryotic messenger RNA by means of the polymerase

chain reaction. Science (New York, N.Y.), 257(5072), pp.967 – 71. Available at:

http://www.ncbi.nlm.nih.gov/pubmed/1354393 [Accessed April 9, 2015].

23. Luan, J.-B. et al., (2011). Global analysis of the transcriptional response of whitefly to tomato yellow leaf curl

China virus reveals the relationship of coevolved adaptations. Journal of virology, 85(7), pp.3330 – 3340.

24. Mahadav, A. et al., (2008). Parasitization by the wasp Eretmocerus mundus induces transcription of genes related

to immune response and symbiotic bacteria proliferation in the whitefly Bemisia tabaci. BMC genomics, 9(1),

p.342. Available at: http://www.biomedcentral.com/1471-2164/9/342 [Accessed March 19, 2015].

25. Morin, S. et al., (2000). The GroEL protein of the whitefly Bemisia tabaci interacts with the coat protein of

transmissible and nontransmissible begomoviruses in the yeast two-hybrid system. Virology, 276(2), pp.404 – 16.

Available at: http://www.sciencedirect.com/science/article/pii/S004268220090549X [Accessed February 10,

2015].

26. Rahman, M. et al., (2013). Science Identification of differentially expressed genes in gauze-exposed omentum of

dogs using differential display RT-PCR. , 14, pp.167 – 173.

27. Robinson, R., (2008). For mammals, loss of yolk and gain of milk went hand in hand. PLoS biology, 6(3), p.e77.

Available at: http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.0060077 [Accessed February

26, 2015].

28. Schrick, K. et al., (2004). START lipid/sterol-binding domains are amplified in plants and are predominantly

associated with homeodomain transcription factors. Genome Biology, 5(6), p.R41. Available at:

http://genomebiology.com/2004/5/6/R41 [Accessed March 17, 2015].

29. Shadmany, M., Omar, D. & Muhamad, R., (2013). First Report of Bemisia tabaci (Hemiptera: Aleyrodidae)

Biotype Q in Malaysia. Florida Entomologist , 96(1), pp.280 – 282. Available at:

http://dx.doi.org/10.1653/024.096.0147 [Accessed March 19, 2015].

30. Shu, Y. et al., (2009). Molecular characterization and expression pattern of Spodoptera litura (Lepidoptera:

Noctuidae) vitellogenin, and its response to lead stress. Journal of insect physiology, 55(7), pp.608 – 16. Available

at: http://www.ncbi.nlm.nih.gov/pubmed/19482134 [Accessed March 13, 2015].

31. Sieglaff, D.H., Duncan, K.A. & Brown, M.R., (2005). Expression of genes encoding proteins involved in

ecdysteroidogenesis in the female mosquito, Aedes aegypti. Insect biochemistry and molecular biology, 35(5),

pp.471 – 90. Available at: http://www.sciencedirect.com/science/article/pii/S0965174805000391 [Accessed March

19, 2015].

32.

Soccio, R.E. & Breslow, J.L., (2003). StAR-related Lipid Transfer (START) Proteins: Mediators of Intracellular

Lipid Metabolism. Journal of Biological Chemistry, 278(25), pp.22183 – 22186. Available at:



10/18



http://www.jbc.org/content/278/25/22183.full?ijkey=2d0cccaa1c3f4623c6825b26801575bb4a06369b&keytype2=

tf_ipsecsha [Accessed March 17, 2015].

33. Su, Q. et al., (2013). Insect symbiont facilitates vector acquisition, retention, and transmission of plant virus.

Scientific reports, 3, p.1367. Available at:

http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=3586701&tool=pmcentrez&rendertype=abstract.

34. Teixeira, L., Ferreira, A. & Ashburner, M., (2008). The bacterial symbiont Wolbachia induces resistance to RNA

viral infections in Drosophila melanogaster. PLoS biology, 6(12), p.e2. Available at:

http://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.1000002 [Accessed February 13, 2015].

35. Tufail, M. & Takeda, M., (2009). Insect vitellogenin/lipophorin receptors: molecular structures, role in oogenesis,

and regulatory mechanisms. Journal of insect physiology, 55(2), pp.87 – 103. Available at:

http://www.ncbi.nlm.nih.gov/pubmed/19071131 [Accessed February 26, 2015].

Venkata, B.P. & Schrick, K., (2006). START domains in lipid/sterol transfer and signaling in plants. Symposium A

Quarterly Journal In Modern Foreign Literatures.

Figure 1: Gel Electrophoresis of ACP - differential Display analysis of viruliferous and non-viruliferous insects

using anchored Primers ACP 2-dt with 4 Arbitrary primers ACP14, ACP20, ACP22, ACP32 , M is 100bp VivantisMarker, 1: is Viruliferous and 2: is Non Viruliferous. Arrows Indicate a Number of different Fragments. The Long

Arrow shows the direction of Bands Selection (from the Top to the Bottom of the gel)

ACP14 -1 (605 bp)

GTCTACCAGGCATTCGCTTCATGGGGGGCAAGTCGGCAATTTTTAAAGAATAGCTTACGCTTGAATA

TTCAGTCCATGGGTGTTGAGATTGGTGGCTCCATCGATCAATATTCTACTCTACACGTATATTACTAAGACCT

Figure 2: Representing the Sequences of Selected Fragments Resulted from the Combination of Anchored

Primer dt-acp2 with Arbitrary Primer ACP14

ATTATAAGGACCTAGTTTTAAATCTCACCTAATATTGTAGTCATAATCATATATATTAATCAGTGTGC


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TATTCTTAACTTTAAATTTTAAGACCATCTGAACATCACAAAGGCAGAAATGTGAAGTTTATTCTTGAGTTTT

CAAAGTTGTACATAATTTTGATATTTTATCTATCTTAAACATATTGTTGAGTAAGTCTCTTAATTTTATCCTAT

ATGTGTTTGTGTATTTGTTGCTACATGTTTAATGTTTATCTTCTATTCTATCTCAGTATATTTATTCACAAACAC

TTCACAAAGGATTATGAAGTCATCAAAATGTCTCCCGAAGATTCAGTGATGACTAGAATTTCCCCCAGAAATAATAATGGAAGATTAGAAAAGTGAAGTAAAATGAACGGTTAACACCGACGAAAATGTCTGTTGTCTTGTCA

CAAGAACTTCAAATAAAAGTCATTTTACTGTG

ACP14 -2 (214 bp)

TGTCTACCAGGCATTCGCTTCATGGGGGGCAAGTCGGGCGGCATCATACAGGTTGGCAAGCGGCGC

ACGGGTGAGTAATACATGTAAATATACCTAAAAGTGGGGAATAACGTACGGAAACGTACGCTAATACCGCA

TAATTATTACGAGATAAAGCAGGGGCTTGATAAAAAAAAAAAAAAAAAACCCCCATCGTAGTCGCAGCATT

CACAGA

ACP14 -3 (398 bp)

GTCTACCAGGCATTCGCTTCATGGGGGGCAAGTCGGGAAAATGAAGGCTCCTAGCTCAAAGGGGAA

AATTGAAAACCTTGGGGTCAGTTCTAAAATGAACGGTAAGAATTTTTCATTTTCCTTTCATTTTGTCAATGGA

TTGTTGGTACAGAAAAAGAGACTTGCGATTTTTTAGCTTGGTAGAACCTGAAAGGGCAAGGGGATTTTCCTG

AATACGCTCTGAATGAATTAGTATCAGCAATTTCCAAGGTAAAAAATTATTGAGATAGCGCAATTAGTTAAA

GTTGATGGTTGCCTTCTTTCTTTGACTGAATAGACATTAGTTAGACGTCTTACAAAAAAAAAAAAAAAAAAA

CCCCCATCGTAGTCGCAGCATTCACAGAATCACTAGTGAATTC

ACP14-4(479bp)

TCTACCAGGCATTCGCTTCATGGGGGGCAAGTCGAGCGGCATCATACAGGTTGGCAAGCGGCGCAC

GGGTGAGTAATACATGTAAATATACCTAAAAGTGGGGAATAACGTACGGAAACGTACGCTAATACCGCATA

ATTATTACGAGATAAAGCAGGGGCTTGATAAAAAAAATCAACCTTGCGCTTTTAGAAAATTACATGCCGGAT

TAGCTAGTTGGTAGAGTAAAAGCCTACCAAGGTAACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACA

CTGGGACTGAGAAAAGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGGAACCC

TGATCCAGTCATGCCGCGTGTGTGAAGAAGGCCTTTGGGTTGTAAAGCACTTTCAGCGAAGAAGAAAAAAA

AAAAAAAAAAAACCCCCATCGTAGTCGCAGCATTCACAGAATCACTAGTGAATTCGC

ACP14 -5 (496bp)

GTCTACCAGGCATTCGCTTCATGGGGGGCAAGTCGGCTTCGAACATTATCTCAAACCATCGTCCGAT

CAGCAAATGATTTACTAGTAAGATTCTCTATTACCTCTAATTGTTCCTTCTTAACACCCAACAGAGCAAAATC

ATTATTTTGTGTTGACAATCTCAAGAGCACCTTATCAAAACTGAAGATGCGAATTAAAGGGGATTTAGCACA

ATTTTCACAAGCGGGAAAGAAACCGGTGACTGCTAACCCAGTGATAAAATTTGTTTGCGTTTTTATCCCTGA

GACAACTTTTGTAAAAATGTTTAATTTTTTTAATTACTCATCATGGAGAAGCCAGCTTCGTGTAAATGAAATG

AACACTGACTGTTTTTCCCACTTTTTTTAAAGTCAGCAAGAAAATTTGATCGATGTTAATGAAATGGAATATT

TAATTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCCCCCATCGTAGTCGCAGCATTCACAGA

Figure 2: cont.,



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ACP14 -6 (242bp)

ATTGTCTACCAGGCATTCGCTTCATGGGGGGCAAGTCGGCAGAAGAAAAAATTCAAAATGAATTGA

TGGAAATGAGAAGACGAGAGGAAGAGCTGAGACGACAGAGAGCATTCTCTTTGGCTAGATCACAACCAAAT

TTATTAAGTCTAATTGACGGTGAGGGGGAAATTGTAGATAACGAAAAAAAAAAAAAAAAACCCCCATCGTA

GTCGCAGCATTCACAGAATCACTAGTGAATTCGC

ACP14 -7 (245bp)

GTGTTCAAGCCTCGTGCTGGCGACAGAGGATCCGTAGGAGCTGTTGTCGTCAGAGGAGCTAGAGTC

AGAGGAGCTGGAGGAGCTAACTGGAAGGAAGAGCTGGAGGAGCTGGAGGAGCTGGAAGAGTCGGAGGAGT

CAGAGGAGCTGCTGGAGGAGGAGGATCCAGATCCGTGGTGGCTGTTCAAACAGTCCATGAAATGTCCCGCG

GTGGTCCGTTTTCATCGCGGATTCTCTCTAGTATGGCT

Figure 2: cont.,

ACP20 -1 (541bp)

CTGTGAATGCTGCGACTACGATGGGGGTTTTTTTTTTTTTTTTTTTTTATTTTTTCTAACTACATAAAT

AAAATATTTAAACAAATAAATAAATAATAAATAGTTAACATACTTAAATTCATTATAAAAGTGAAGATGAGA

AATGAGTGAATAATTTTCACTGTTTCTCCAGGAGGCGAAAGTTGGGAGGTAGGAAAAGAAGTGCCAAAGCT

CTCAGGAAATCTGCCGGTAGGACAGGTTTTAAGGTCAAGCAAAGGAAGGAGGGGTGGGTATTAGTTTCAAA

GGAGGCATGAGATTGGGAAGCATCATTTTTGGTCCATCAGCATCATTTTACAGGTCCAGCATTACTTTCTGTG

ACCACAATTCCATTGCGATCAATGAAAATATTTTCAAGATCACAATTTCCATCATCATTTTTCGTTGTTCATTG

TTGTTAGAAGCATCATTTTTTTATATTTTCTGAAGCATAGTACAGAGCAGTGAACTACTCTTCTTCATCGACG

CCAACGTCCCCCCATGAAGCGAATGCCTGGTAGACA

ACP20 -2 (359 bp)

GTCTACCAGGCATTCGCTTCATGGGGGGACGTTGGCGCATGTGTCGTGCAGTTCAACTGGGAGAGAG

AGAGAGAATTTCTGATGATGAATTATTCAGGCATTCACGCGAATTTGAAAGGATTTACCGGTTACAGAGTAC

AATATCAGCCCCACTTTGCTCTCACTCAGAACTACAGTGCCAGTCTCAGGGCTCTCAAAAGAAAGATCATCA

AAAAAGTAGTGTGTCCAATGAGACCATCTGCAATTGTTCTCGTTGTAGAAATTCGTCAGATGACGGTTGGGT

AGAATTTTATAGAGGTGATAGTTTTATTGTATGAAAAAAAAAAAAAAAACCCCCATCGTAGTCGCAGCATTC

ACAG

Figure 3: Representing the Sequences of Selected Fragments Resulted from the Combination of Anchored Primer

dt-acp2 with Arbitrary Primer, ACP20

ACP22 -1 (438bp)

TGTGAATGCTGCGACTACGATGGGGGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAAGTAATTAACAA

CACACGTGTATTTTGATCAACTGAGGAACATGGGAAGAAAACAAAACAACTATCTTTGGTGACTTACAAATG

Figure4: Representing the Sequences of Selected Fragments resulted from the Combination of Anchored

Primer dt-acp2 with Arbitrary Primer ACP22


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CGATGATGTTGCAACACTTAGTGGTGGTAGCTTTCGACGGGAGGGCAGACCTCTTCGACGATGACGG

CTTGCTTGGCGGGCTCTTCGTGCACCTGGGGGTGTTCGTACTCGTCGACAACGACGGGCTTCTGGGTGTAGGC

GTGGAGGACAGCGCTCTGGGTGTGCTTGACTACGTAGGGTTGTCCGTAGTAGTGGAAGACAGAGGGAGCAG

AGCGGTGCTCGATCACCTGGGGTTCACGGTGGACTCCGTAGGTGTATGGCACGGGTACCCCCCATGAAGCGAATGCCTGGTAGAC

ACP22 -2 (245 bp)

AAGCTTCGGCATAGATGAAACGACGATCACCGGGATGATGGACAGGGAAACCTGGATGAATGGAG

GCGAAGCGATAGAGAAAGGGTTTGCGGATGCCCTCCTGCCCGCAGACAGCACGCAGCAGGATAACGATTCG

CCCATCGCCGCCCTGAGAAAACTGGAGGCACTCCTGGCAAAAGCCAATACGCCCCGTGCTGAACGACGACG

ATTATTGAACGCCTTACGCGGTGATATGCCGAAGCTTA

Figure 4: cont.,

ACP32 -1(910bp)

GTCTACCAGGCATTCGCTTCATGGGGGGCGCTACTCCTCATCGTATCAGTACCCTAAGGAACAAGAC

GACATTGTTCCGTTCCCTTCATCCTGGGTGTTAGTAAAACAGTAGCCGGTTAAAGCGAAATGCTTTTAAAGA

AGGAGTAGTATCATATTTAGTAGGAGGCACAGTAGGCAGTTAGGACGGAGAAGCAACTAATCTAGCGGAGA

AAACTTGATTTGTTGTTGTTAGTACTAAAGCTTCCTCAAAGGTAATTATTGGGTTCTCCTTGAAAGAATTAGA

ATGTTTCGTTTGAATTTATGCTGATATCTTAACGAGTTCAGTCTGTTTCGTTGAAACTCCTGAAAATCAAGAG

AGCAGTCTTCATCTGGATGATAGCACTAATATGGGAATTGGTATTGGGGTGCCTCATCATGAACTAAAACAG

GGTTGCCTCTGCCTCTCCTCATGAATTGAAGGTCCCTGAAATGGCAAAAAAAAAAAAAAATTGGAAAAAAA

AAACCCCAATCCCCCCAATAAAATGGCGTTTTGCCCCTTTTTCAAACCTCCCCTTTCCAGGGAGTCCAATTCC

GGGGGAATTGGGTTAACCCTAACATTAGGGCTCGGAACCGTTTTCTTGGAAATTCCGATTCAAATTCGGTGG

AACCTAAAAATTTTTTTTTCCCCCCCCCCCCCCCCGTTTAAATTTTTTTCAGGGTTTTAAAAAACTTTCCCCGG

GGTCCAAAAAAAAAAAAAAAACCCCCCCCTTTTTTTTTCCCAAAATTTCCAAAAAAACCCTTTGGGAATTTTC

CCCGGCCCCCCGGGGGGGGCCCCAAATTTGGGGGAAAACCCCCCCCCCCCCCGTTTTGGAAAAACCCCCTTG

GGGTTTTTTTTTTTTGGGGGGCCCAAAAAAAAAAGGGGGGGGGGGAA

ACP-32-2 (203BP)

GGGGGGGGGGCTCAGGGCCCGGCCGCCCTGGGGGGCCGTGGACATTAGGTTGCTCGGACAGCGACA

TGGCTGAATGGGGCTTAATAATACCCATCGTGTCAATACGCTTGGGAACAAGACAAGGTGCGATCTGTGTCC

ATTATGCTGGAGCACTTGTAATGCGCGCCGCCTGTAGATCAAACCCGCACTTATGGAACGAGAAG

ACP32-3 (185BP)

GCTGTGAATGCTGCGACTACGATGGGGGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAA

ATTACAAGAATTTTTTTTTAATTATTCAATTAAAAATAATAATATAAATCTATTTAAAAGGGATAAATCTTGA

AAAGGGAGTAGCGCCCCCCATGAAGCGAATGCCTGGTAAACA

Figure5: Representing the Sequences of Selected Fragments Resulted from the Combination of Anchored Primer

dt-acp2 with Arbitrary Primer ACP32



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Figure 6: qRT-PCR Curves of Representative Sample Replicates from Viruliferous and Non-viruliferous Whiteflies

Replicates, HSP90 Gene is at the Same Level in Both the Viruliferous and Non-viruliferous Whiteflies, A:

Amplification Curve of Candidatus Porti era 16S Ribosomal RNA gene, B: Amplification Curve of stAR-related

Lipid transfer protein, C: amplification curve of peptidase gene, D: amplification curve of Primary endosymbiont

16S Ribosomal RNA gene, E:amplification curve of vitellogenin Gene

Figure 7: Δct of Viruliferous and Non-viruliferous whitefly of Expression of Endosymbiont of Bemisia tabaci 16S

Ribosomal RNA Gene (16Sp.endo), Candidatus Porti era aleyrodidarum 16S Ribosomal RNA(16Scan), Steroidogenic

Acute Regulatory protein (StAR)-related Lipid Transfer (START) Domain, Peptidase and vitellogenin. Reactions

were normalized to a Stable Housekeeping Gene (HSP90). All Reactions were performed in Triplicate, and the

Average Δct is shown


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Figure 8: ΔΔct between Viruliferous and Non-viruliferous Whitefly of Expression of Endosymbiont of Bemisia

tabaci 16S Ribosomal RNA gene(16Sp.endo), Candidatus Porti era aleyrodidarum 16S ribosomal RNA(16Scan),

Steroidogenic Acute Regulatory Protein (StAR)-related Lipid Transfer (START) domain, Peptidase and

Vitellogenin. Reactions were normalized to a Stable Housekeeping Gene (HSP90). All Reactions were performed in

Triplicate, and the Average ΔΔct is shown

Figure 9: Real-time PCR Analyses for differentially Expressed Endosymbiont of Bemisia tabaci 16S ribosomal RNAgene, Candidatus portiera aleyrodidarum 16S Ribosomal RNA, Steroidogenic Acute Regulatory Protein (StAR)-

Related Lipid Transfer (START) domain, Peptidase and Vitellogenin in Viruliferous Whitefly. Reactions were

normalized to a Stable Housekeeping Gene (HSP90), and fold changes were calculated relative to the Non-

viruliferous one. All Reactions were Performed in Triplicate, and the Average fold Change is Shown



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Table 1: Primer Sequences Used for cDNA Synthesis and Annealing Control Primer (ACP)-based PCR

PurposePrimer

NamePrimer Sequence Ref.

First-strand

cDNAsynthesis

dT-

ACP1 5´-CTGTGAATGCTGCGACTACGATIIIII(T)18-3´

(Rahman

et al.

2013)

Reverse

primer for

PCR

dT-

ACP25´-CTGTGAATGCTGCGACTACGATIIIII(T)15-3´

Forward

primer

(arbitrary

primer) forPCR

ACP14

ACP20

ACP22

ACP32

5´-GTCTACCAGGCATTCGCTTCATIIIIIGCAAGTCGGC-3´

5´-GTCTACCAGGCATTCGCTTCATIIIIIGACGTTGGCG-3´

5´-GTCTACCAGGCATTCGCTTCATIIIIIGTACCCGTGC-3´

5´-GTCTACCAGGCATTCGCTTCATIIIIIGCGCTACTCC-3´

Table 2: Representing Expression Pattern of differentially Expressed Bands

CloneName

Expression Pattern RegulationType

Clean Infected

1 Acp14 -1 ++ +- Down

2 ACP14-2 + - Down

3 ACP14-3 ++++ + Down

4 ACP14-4 ++ + Down

5 ACP14-5 - + Up

6 ACP14-6 +- ++ Up

7 ACP14-7 - +- Up

8 ACP20-1 + - Down

9 ACP20-2 - +- Up

10 ACP22-1 + - Down11 ACP22-2 - + up

12 ACP32-1 + +- Down

13 ACP32-2 + - Down

14 ACP32-3 - +- Up

Table 3: List of Primers Used for Quantitative Reverse Transcription – Real Time PCR for Validation of

differentially Expressed Genes

Fragment no. Primer sequenceProducts size

(bp)

HSP 90Forward ATCGCCAAATCTGGAACTAAAGC

90bpReverse GTGTTTTGAGACGACTGTGACGGTG

ACP14-2Forward TCATACAGGTTGGCAAGCG

77bpReverse TTTCCGTACGTTATTCCCCAC

ACP14-4Forward GCCGGATTAGCTAGTTGGTAG

95bpReverse CCTTTTCTCAGTCCCAGTGTG

ACP14-7Forward TCCGACTCTTCCAGCTCC

90bpReverse GTTGTCGTCAGAGGAGCTAG

ACP20-2Forward CAGGATTCGCTTCATGGGGGGA

123Reverse GGGGTGATATTGTACTGT

ACP22-2Forward GCATAGATGAAACGACGAT

110Reverse CTCAGGGCGGCGATGGGCGA


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Table 4: Differentially Expressed Gene Transcripts Showing Sequence Similarities with known genes

Clone Homology Accession No. Species

Fragme

nt

size

E-

value

Expressio

n

pattern

Fold

change

ACP-14-1 cDNA library ofTYLCV infected

whitefly

EE602294.1 Bemisia tabaci 605 bp 0.0 Down ---------

ACP-14-2Primaryendosymbiont16

S ribosomal RNA geneAF400460.1 Bemisia tabaci 214 bp 1e

-67 Down -5.88

ACP-14-4Candidatus Portiera

16S ribosomal RNA NR_102830.1 Bemisia tabaci 479 bp 0.0

Down-100

ACP-14-7 vitellogenin Bemisia tabaci 245bp 6e-41

Up 9.25

ACP-20-2stAR-related lipid

transfer protein

XP_001943914.

1

Acyrthosiphon

pisum359 bp 0.043 Up 2.44

ACP-22-1Cdna library of TYLCV

infected whiteflyEE602081.1 Bemisia tabaci 438 2e-

158 Down ---------

ACP-22-2 peptidase WP_041387620

Plautia stali

symbiont 245 3e-19 UP 4.92

ACP-32-2

cDNA library of

TYLCV infected

whitefly

EE599909.1 Bemisia tabaci 910 3e-66

Down ----------

Table 5: Quantitative Real Time PCR Estimation of Expression of Endosymbiont of Bemisia tabaci 16S Ribosomal

RNA gene, Candidatus Porti era aleyrodidarum 16S Ribosomal RNA and Steroidogenic Acute Regulatory Protein

(StAR)-related Lipid Transfer (START) Domain, Peptidase and Vitellogenin in Case of Viruliferous and Non-

Viruliferous Whiteflies

http://www.tjprc.org/http://www.tjprc.org/http://www.ncbi.nlm.nih.gov/nucleotide/113017961?report=genbank&log$=nucltop&blast_rank=1&RID=EVKP2JE501Rhttp://www.ncbi.nlm.nih.gov/nucleotide/18029912?report=genbank&log$=nucltop&blast_rank=1&RID=XN8T4CVZ01Rhttp://www.ncbi.nlm.nih.gov/nucleotide/507148023?report=genbank&log$=nucltop&blast_rank=1&RID=XNKCV8KR014http://www.ncbi.nlm.nih.gov/protein/193615447?report=genbank&log$=prottop&blast_rank=2&RID=FVJ213G0015http://www.ncbi.nlm.nih.gov/protein/193615447?report=genbank&log$=prottop&blast_rank=2&RID=FVJ213G0015http://www.ncbi.nlm.nih.gov/nucleotide/113020438?report=genbank&log$=nucltop&blast_rank=1&RID=J5JND6MK01Rhttp://www.ncbi.nlm.nih.gov/nucleotide/113015217?report=genbank&log$=nucltop&blast_rank=1&RID=J57SPKTS016http://www.ncbi.nlm.nih.gov/nucleotide/113015217?report=genbank&log$=nucltop&blast_rank=1&RID=J57SPKTS016http://www.ncbi.nlm.nih.gov/nucleotide/113020438?report=genbank&log$=nucltop&blast_rank=1&RID=J5JND6MK01Rhttp://www.ncbi.nlm.nih.gov/protein/193615447?report=genbank&log$=prottop&blast_rank=2&RID=FVJ213G0015http://www.ncbi.nlm.nih.gov/protein/193615447?report=genbank&log$=prottop&blast_rank=2&RID=FVJ213G0015http://www.ncbi.nlm.nih.gov/nucleotide/507148023?report=genbank&log$=nucltop&blast_rank=1&RID=XNKCV8KR014http://www.ncbi.nlm.nih.gov/nucleotide/18029912?report=genbank&log$=nucltop&blast_rank=1&RID=XN8T4CVZ01Rhttp://www.ncbi.nlm.nih.gov/nucleotide/113017961?report=genbank&log$=nucltop&blast_rank=1&RID=EVKP2JE501Rhttp://www.tjprc.org/


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Documents

2. Biotech - IJBTR -Identification of differentially expressed genes - Inas Farouk Fahmy.pdf