Plant Pathology Department
First Advisor
Amit Mitra
Committee Members
Loren Giesler, Ismail Dweikat
Date of this Version
8-2024
Document Type
Thesis
Citation
A thesis presented to the faculty of the Graduate College at the University of Nebraska in partial fulfillment of requirements for the degree of Master of Science
Major: Plant Pathology
Under the supervision of Professor Amit Mitra
Lincoln, Nebraska, August 2024
Abstract
Wheat (Triticum aestivum L.) is the principal cereal grown in the United States, whose production ranks third in the world. Bacterial leaf streak (BLS) disease caused by Xanthomonas translucens pv. undulosa (xtu) is an emerging threat to wheat production in the Great Plains region of the United States of America. In this study, we engineered sequences encoding several plant-based antimicrobial peptides (AMP) were engineered into the WSMV genome and examined their effect on BLS disease in wheat infected by WSMV harboring AMP sequences. Out of nine AMPs, wheat expressing Ace-AMP1, PDF13, TK-AMP-D1, and TK-AMP-D3 produced resistance against the pathogen. Expression of dual AMPs in wheat produced lesions of 1.7, 1.4 , and 1.7 cm, respectively, compared to wheat expressing single AMPs (1.8-2.6 cm) and WSMV-infected (8.1 cm) wheat at 21 dpbi. This data suggest that Ace-AMP1, PDF13, TK-AMP-D1, and TK-AMP-D3 either singly- or doubly in wheat significantly reduced the lesion size of BLS disease in wheat.
Wheat streak mosaic virus (WSMV; genus Tritimovirus; family Potyviridae) is an economically important virus on wheat in the Great Plains. In this study, we examined the content and composition of WSMV virions through mass spectrometry and Western blot using virions purified through 10-40% sucrose density gradient and cesium chloride centrifugation. Mass spectrometric analysis of protein composition of purified WSMV virion preparation indicated that coat protein comprises 55-80% of total proteins in virion preparations. Surprisingly, virion preparations also contained four virus-encoded proteins as components of virions: HC-Pro (3-8%), CI (0.8%), P1 (0.8%),and NIa-VPg (0.2-0.4%).Western blot analysis confirmed the presence of CP, HC-Pro, and P1 as components of WSMV virions. Previously, we found that GFP was expressed as a fusion protein with HC-Pro in a GFP-tagged WSMV (WSMV-GFP)-infected wheat leaves. Examination of purified virions from WSMV-GFP-infected wheat leaves revealed that virions also contained GFP as GFP-HC-Pro fusion protein. Additionally, purified virions from WSMV-GFP-infected leaves elicited fluorescence under a confocal microscope, suggesting that functional GFP was incorporated into WSMV virions as GFP-HC-Pro fusion protein. These data suggest the expression of specialty products on the surface of WSMV virions can be achieved by fusing the novel amino acid sequences to HC-Pro. Taken together, our data revealed that WSMV is unusual in incorporating four accessory proteins (HC-Pro, CI, P1, and NIa-VPg) along with CP (major protein) as components of virion.
Advisor: Amit Mitra
Comments
Copyright 2024, Sourav Pal. Used by permission