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Department of Microbial molecular biology, Agriculture Genetic Engineering Research Institute, Giza, Egypt
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Department of Molecular Biology, Agriculture Genetic Engineering Research Institute, Giza, Egypt
Department of Nucleic acid Structure and Function, Agriculture Genetic Engineering Research Institute, Giza, Egypt
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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 downregulation, 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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Inasf. Fahmy, Asmaa F. Darweesh, Mohmed A. El-Satar, Rania M. Abou-Ali, Ahmed H. M. Elwahy
(Anon 2007).
Begomovirus is the largest genus among the four genera of Geminiviridae, whereas these genera are classified according to
their genome organization and host range (Hanssen et al. 2010). Begomoviruses passage through their insect vector in a
circulative persistent manner, from the gut lumen into the hemolymph and finally routing to the salivary glands, from
which these viruses are secreted again into the plant host during insect sap feeding(Czosnek & Ghanim 2002; Hogenhout et
al. 2008). During this process, a homolog protein GroEL produced by the B. tabaci endosymbiotic bacteria and the
whitefly BtHSP16 protein have reportedly been involved in the viral transmission(Morin et al. 2000).Bemisia tabaci is the
host of a wide range of bacterial symbionts. In the gut of B. tabaci, these bacteria live within bacteriocyte cells that are
transmitted directly from the parent to oocytes. These bacteria is divided into two types: primary symbionts (P-symbionts)
and secondary symbionts (S-symbionts) (Baumann 2005).
Differential display reverse transcription polymerase chain reaction (DD RT-PCR) is a useful technique for studying
different sets of genes expressed in tissues(Kao et al. 2003). It is possible to identify new and unexpected changes in
transcription, making the non-biased gene screening feasible and does not require any prior mRNA genomic information
(Liang et al. 1993).The discovery and characterization of the differentially expressed genes between the viruliferous and
non-viruliferous whitefly would be a useful tool for the identification of the vector-virus inter-relations that could be
exploited to generate TYLCV resistant lines.
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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
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Inasf. Fahmy, Asmaa F. Darweesh, Mohmed A. El-Satar, Rania M. Abou-Ali, Ahmed H. M. Elwahy
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-66
and 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 realtime 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 calculated
using the 2CTmethod. 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;BTTYLCV-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, hostplant 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
Impact Factor (JCC): 3.1245
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survival but may play important roles in host biology and evolution (Himler et al. 2011). Whiteflies harbor an obligatory
symbiotic bacteriumCandidatus 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 whitefly
vector (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 hosts 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 upregulated 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 affect Vg production.
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Inasf. Fahmy, Asmaa F. Darweesh, Mohmed A. El-Satar, Rania M. Abou-Ali, Ahmed H. M. Elwahy
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 disarm
viruses 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 downregulated 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 upregulation 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 RACEtechnique 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
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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.
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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 Vivantis
Marker, 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
19
TATTCTTAACTTTAAATTTTAAGACCATCTGAACATCACAAAGGCAGAAATGTGAAGTTTATTCTTGAGTTTT
CAAAGTTGTACATAATTTTGATATTTTATCTATCTTAAACATATTGTTGAGTAAGTCTCTTAATTTTATCCTAT
ATGTGTTTGTGTATTTGTTGCTACATGTTTAATGTTTATCTTCTATTCTATCTCAGTATATTTATTCACAAACAC
TTCACAAAGGATTATGAAGTCATCAAAATGTCTCCCGAAGATTCAGTGATGACTAGAATTTCCCCCAGAAAT
AATAATGGAAGATTAGAAAAGTGAAGTAAAATGAACGGTTAACACCGACGAAAATGTCTGTTGTCTTGTCA
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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Inasf. Fahmy, Asmaa F. Darweesh, Mohmed A. El-Satar, Rania M. Abou-Ali, Ahmed H. M. Elwahy
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
Impact Factor (JCC): 3.1245
21
CGATGATGTTGCAACACTTAGTGGTGGTAGCTTTCGACGGGAGGGCAGACCTCTTCGACGATGACGG
CTTGCTTGGCGGGCTCTTCGTGCACCTGGGGGTGTTCGTACTCGTCGACAACGACGGGCTTCTGGGTGTAGGC
GTGGAGGACAGCGCTCTGGGTGTGCTTGACTACGTAGGGTTGTCCGTAGTAGTGGAAGACAGAGGGAGCAG
AGCGGTGCTCGATCACCTGGGGTTCACGGTGGACTCCGTAGGTGTATGGCACGGGTACCCCCCATGAAGCGA
ATGCCTGGTAGAC
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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Inasf. Fahmy, Asmaa F. Darweesh, Mohmed A. El-Satar, Rania M. Abou-Ali, Ahmed H. M. Elwahy
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 Portiera 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: ct of Viruliferous and Non-viruliferous whitefly of Expression of Endosymbiont of Bemisia tabaci 16S
Ribosomal RNA Gene (16Sp.endo), Candidatus Portiera 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 ct is shown
23
Figure 9: Real-time PCR Analyses for differentially Expressed Endosymbiont of Bemisia tabaci 16S ribosomal RNA
gene, 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 Nonviruliferous one. All Reactions were Performed in Triplicate, and the Average fold Change is Shown
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Inasf. Fahmy, Asmaa F. Darweesh, Mohmed A. El-Satar, Rania M. Abou-Ali, Ahmed H. M. Elwahy
Table 1: Primer Sequences Used for cDNA Synthesis and Annealing Control Primer (ACP)-based PCR
Purpose
First-strand
cDNA
synthesis
Reverse
primer for
PCR
Forward
primer
(arbitrary
primer) for
PCR
Primer
Name
Primer Sequence
Ref.
dTACP1
5-CTGTGAATGCTGCGACTACGATIIIII(T)18-3
dTACP2
5-CTGTGAATGCTGCGACTACGATIIIII(T)15-3
ACP14
ACP20
ACP22
ACP32
5-GTCTACCAGGCATTCGCTTCATIIIIIGCAAGTCGGC-3
5-GTCTACCAGGCATTCGCTTCATIIIIIGACGTTGGCG-3
5-GTCTACCAGGCATTCGCTTCATIIIIIGTACCCGTGC-3
5-GTCTACCAGGCATTCGCTTCATIIIIIGCGCTACTCC-3
(Rahman
et al.
2013)
Acp14 -1
ACP14-2
ACP14-3
ACP14-4
ACP14-5
ACP14-6
ACP14-7
ACP20-1
ACP20-2
ACP22-1
ACP22-2
ACP32-1
ACP32-2
ACP32-3
Expression Pattern
Clean
++
+
++++
++
++
+
+
+
-
Infected
++
+
+
++
+++
++-
Regulation
Type
Down
Down
Down
Down
Up
Up
Up
Down
Up
Down
up
Down
Down
Up
Table 3: List of Primers Used for Quantitative Reverse Transcription Real Time PCR for Validation of
differentially Expressed Genes
Fragment no.
HSP 90
ACP14-2
ACP14-4
ACP14-7
ACP20-2
ACP22-2
Primer sequence
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
Forward
Reverse
ATCGCCAAATCTGGAACTAAAGC
GTGTTTTGAGACGACTGTGACGGTG
TCATACAGGTTGGCAAGCG
TTTCCGTACGTTATTCCCCAC
GCCGGATTAGCTAGTTGGTAG
CCTTTTCTCAGTCCCAGTGTG
TCCGACTCTTCCAGCTCC
GTTGTCGTCAGAGGAGCTAG
CAGGATTCGCTTCATGGGGGGA
GGGGTGATATTGTACTGT
GCATAGATGAAACGACGAT
CTCAGGGCGGCGATGGGCGA
Products size
(bp)
90bp
77bp
95bp
90bp
123
110
25
Table 4: Differentially Expressed Gene Transcripts Showing Sequence Similarities with known genes
Clone
ACP-14-1
ACP-14-2
ACP-14-4
ACP-14-7
ACP-20-2
ACP-22-1
Homology
cDNA
library
of
TYLCV
infected
whitefly
Primaryendosymbiont16
S ribosomal RNA gene
Candidatus
Portiera
16S ribosomal RNA
vitellogenin
stAR-related
lipid
transfer protein
Cdna library of TYLCV
infected whitefly
ACP-22-2
peptidase
ACP-32-2
cDNA
TYLCV
whitefly
library
of
infected
Accession No.
Species
Fragme
nt
size
Evalue
Expressio
n
pattern
Fold
change
EE602294.1
Bemisia tabaci
605 bp
0.0
Down
---------
AF400460.1
Bemisia tabaci
214 bp
1e-67
Down
-5.88
NR_102830.1
Bemisia tabaci
479 bp
0.0
Bemisia tabaci
Acyrthosiphon
pisum
245bp
6e
XP_001943914.
1
359 bp
EE602081.1
Bemisia tabaci
WP_041387620
Plautia
symbiont
EE599909.1
Bemisia tabaci
stali
-41
Down
-100
Up
9.25
0.043
Up
2.44
438
2e-158
Down
---------
245
3e-19
UP
4.92
910
3e-66
Down
----------
Table 5: Quantitative Real Time PCR Estimation of Expression of Endosymbiont of Bemisia tabaci 16S Ribosomal
RNA gene, Candidatus Portiera aleyrodidarum 16S Ribosomal RNA and Steroidogenic Acute Regulatory Protein
(StAR)-related Lipid Transfer (START) Domain, Peptidase and Vitellogenin in Case of Viruliferous and NonViruliferous Whiteflies
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