Title page for ETD etd-192912649721251

Type of Document

Dissertation

Author

Qusus, Saba J.

Author's Email Address

squsu

URN

etd-192912649721251

Title

Molecular Studies on Soybean Mosaic Virus-Soybean Interations

Degree

PhD

Department

Plant Pathology, Physiology, and Weed Science

Advisory Committee

Advisor Name	Title
Sue A. Tolin	Committee Chair
Carole L. Cramer	none
George H. Lacy	none
Glenn R. Buss	none
Muriel Lederman	none

Keywords

coat protein
genetics
pathogenesis
potyvirus
protoplast

Date of Defense

1997-04-18

Availability

restricted

Abstract

In the U.S., soybean mosaic virus

(SMV) is classified into seven strain

groups, designated G1 to G7, based

on their different responses on resistant

soybean [Glycine max (L.) Merr.]

cultivars. These responses are:

symptomless or resistant (R), necrotic

(N), and mosaic or susceptible (S).

The gene-for-gene model has been

proposed for SMV-soybean

interactions. In the majority of cultivars,

a single dominant gene, Rsv1, confers

both the R and N responses. In the first

part of this study, the coat protein (CP)

genes of two SMV strains, G1 and G6

were isolated, cloned, and sequenced.

Gene isolation was done by reverse

transcription-polymerase chain reaction

(RT-PCR) on partially purified virus

preparation without prior RNA

extraction. Amplified products were

blunt-end ligated into pNoTA/T7

vector and transformed into competent

cells. Sequencing was performed in

both directions on heat-denatured

double-stranded plasmids. The

predicted 265 amino acid sequence of

the CP of G1 and G6 strains were

98.9% identical, with only two amino

acid differences. Correlating the CP

sequences of G1, G2, G6, and G7,

with their virulence on resistant

soybean cultivars indicated that the CP

is not likely to be the R- and/or

N-determinant in the SMV-soybean

system. The second part of the study

involved studying the pathogenesis of

G1, G6, and G7 strains on inoculated

leaves of R, N, and S soybean cultivars

by leaf imprint immunoassay. Results

indicated four types of reactions: i)

susceptible, showing unrestricted

replication and spread; ii) immune,

where no virus was detected; iii)

systemic spread, showing unrestricted

replication but limited spread along the

veins; and iv) restricted replication and

spread, where infection was restricted

to few foci along the veins. Results of

this study indicated that Rsv1-mediated

resistance is a multicomponent type of

resistance that involves both inhibition

of virus replication as well as

cell-to-cell movement. The third part of

the study aimed at investigating

Rsv1-mediated resistance at the

cellular level. For this purpose, an

SMV-soybean protoplast system was

developed. Protoplast isolation was

based on a combined

cellulase-pectolyase Y-23 digestion

and metrizamide-sorbitol gradient

purification protocol. Virus inoculation

of protoplasts was facilitated by either

polyethelene glycol (PEG) or

poly-L-ornithine (PLO), and method of

detection was by Western blotting

using antiserum to whole virus.

Inoculation by PEG was successful, but

results were irreproducible because of

the adverse effect of PEG on

protoplast viability. Inoculation by PLO

was inconclusive because of the high

background from residual inoculum.

Additional research is needed before a

protoplast system can be used to study

the mechanism of Rsv1 resistance to

SMV at the cellular level.

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