Please use this identifier to cite or link to this item: http://prr.hec.gov.pk/jspui/handle/123456789/7333
Title: Analysis of the Potential of Non-Pathogen Derived Resistance Against Begomoviruses
Authors: Ali, Muhammad Yousaf
Keywords: Applied Sciences
Technology
Biotechnology
Issue Date: 2016
Publisher: Pakistan Institute of Engineering and Applied Sciences Nilore, Islamabad, Pakistan
Abstract: Cotton leaf curl disease (CLCuD) is the major threat to cotton crop and is transmitted by whitefly (Bemisia tabaci). Whitefly is also a major pest of cotton crop and significantly damages the crop worldwide. Whitefly bears endosymbionts which live inside gut of insect and endosymbionts produces some protein which may be essential for different metabolic processes. There are some primary endosymbionts which are present in almost all whitefly biotypes but the presence of secondary endosymbionts is dependent on its biotype, ecological distribution and some other unknown factors. These endosymbionts produce a protein GroEL which helps in transmission of cotton leaf curl disease which is a begomoviral disease. There may be co-localization of two or more than two secondary endosymbionts in the same whitefly. The presence of endosymbionts such as Hamiltonella, Rickettsia, Wolbachia, Fritschea, Cardinium and Arsenophonus were tested in whiteflies collected from cotton growing areas of Pakistan and several combinations of endosymbionts were found. Similarly, GroEL gene was cloned and its relationship with already known GroEL was established. Two approaches were used here: these are the control of whitefly through RNAi technology and the expression of GroEL in transgenic plants to generate broad-spectrum resistance. Two RNAi target genes trehalase and trehalose transporter were selected on the basis of their involvement in important physiological pathways in whiteflies. The osmotic pressure of the phloem sap is about five times higher as compared to that in the insect gut. The difference in osmotic pressure forces the fluid to move from phloem into the insect’s gut where it also poses major challenge for the phloem feeders to normalize the pressure by degrading the sucrose content of the phloem sap. Trehalase is present in all tissues and is involved in trehalose transporter metabolism that is it hydrolyzes trehalose transporter into two glucose molecules. In insect trehalase is also found to play important role in functions like flight metabolism, chitin synthesis during molting and cold tolerance. Trehalose transporter is a non-reducing sugar also known as α- D-glucopyranosyl α – D glucopyranoside and is a disaccharide of glucose. Phenotypic manifestation of gene knockdown by dsRNA induced RNAi suggested lowered expression of target genes. Real time qPCR was xv performed in order to estimate transcript levels and the results from expression profiling of whiteflies treated with dsRNA to induce RNAi against the specific targets showed significant success in knocking down expression of the target genes. Bioassays conducted for the target genes showed high mortality rates for dsRNA treated whiteflies, suggesting that the gene knockdown could be an effective way for controlling insect pests like whiteflies. Since multiple begomoviruses and associated satellites are involved in CLCuD, approaches based on the concept of broad-spectrum resistance are essential for effective disease control. GroEL and G5 are two proteins from whitefly endosymbiont and M13 bacteriophage origin respectively. GroEL encapsulates the virus particle when it enters the whitefly and protects the virus from immune system of whitefly as well as prevents viral expression in it. This characteristic of GroEL can be exploited to get resistance against viruses if expressed in plants. G5 is a single stranded DNA binding protein, expression of which in transgenic plants will stop viral expression on its binding with ssDNA. Use of tissue specific promoter is more efficient than constitutive promoters. NSP of BBTV is phloem specific promoter using which, GroEL can be expressed only in phloem tissues, the site for viral entry. To attain broad spectrum resistance, pyramiding of both genes was done. Transgenics of Nicotiana benthamiana for GroEL under constitutive promoter, GroEL under phloem specific promoter, gene pyramid of GroEL and G5 both with their constitutive promoters and gene pyramid of GroEL and G5 in which GroEL under phloem specific and G5 under constitutive promoter were made. In comparison to non-transgenic plants, transgenic plants with double cassette of GroEL under NSP promoter and G5 under 35S promoter showed promising results when challenged against Cotton leaf curl Multan virus (CLCuMuV) along with Cotton leaf curl Multan betasatellite (CLCuMB), Cotton leaf curl Khokhran virus (CLCuKoV) along with Cotton leaf curl Multan betasatellite (CLCuMB) and Pedilenthus leaf curl virus (PedLCV) along with Tobacco leaf curl betasatellite (TbLCB). The data presented here shows that RNAi technology can be effectively used for controlling whiteflies while the expression of GroEL under phloem-specific promoter could be an effective way achieve broad-spectrum resistance.
URI:  http://prr.hec.gov.pk/jspui/handle/123456789//7333
Appears in Collections:PhD Thesis of All Public / Private Sector Universities / DAIs.

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