Comparison of the Symptoms Caused by Three Geminiviruses
in a Common Host
Alex
1
Rajewski ,
2
Dallas ,
2
Hanley-Bowdoin
Mary Beth
and Linda
1Biochemistry, Cell & Molecular Biology Program, Drake University, Des Moines IA 50311
2Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh NC 27606
Symptom Comparison
Abstract
ACMV-Cam
The Geminiviridae are a family of single-stranded DNA plant viruses that
cause severe crop destruction especially in the tropics. Much of the
research into geminivirus replication and resistance is conducted in model
organisms such as Nicotiana benthamiana, which is not the natural host of the
viruses. Model plants are used because of their ease of cultivation and shorter
generation times. Typically, begomoviruses have narrow host ranges and
cannot be compared directly. However, unlike many plant species, N.
benthamiana is susceptible to a variety of begomoviruses and can be used to
compare them in a common host. In this study, we compare the disease
properties of three begomoviruses whose natural hosts include cabbage and
cassava, which represent two families – the Brassicaceae and Euphorbiaceae.
EACMV-UG3
(CaLCuV)
Stunting and Curling
4
3.5
ACMV-Cam
3
2.5
CaLCuV
2
EACMV-UG3
1.5
1
0.5
0
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
19
20
21
22
23
24
Chlorosis
These studies examine Cabbage leaf curl virus (CaLCuV), African cassava
mosaic virus-Cameroon (ACMV-Cam) and East African cassava mosaic virusUganda (EACMV-UG3). CaLCuV infects cabbage, cauliflower and broccoli
fields in the U.S. The two cassava-infecting viruses are part of a disease
complex that causes Cassava Mosaic Disease (CMD) in Africa. CMD is
pandemic across Africa, resulting insufficient food supplies. We infected N.
benthamiana with ACMV-Cam and EACMV-UG3 by bombardment of cloned
viral sequences. CaLCuV was inoculated using Agrobacteria carrying viral
genomic DNA in T-DNA plasmids. The infected plants were monitored for
symptoms and viral DNA accumulation. These studies showed that
CaLCuV and ACMV-Cam caused the most severe symptoms on N.
benthamiana and were similar in severity. EACMV-UG3 symptoms were
significantly milder.
3.5
ACMV-Cam
3
2.5
CaLCuV
EACMV-UG3
2
1.5
1
0.5
0
1
Side-by-Sides: EACMV-UG3 and ACMV-Cam specimens were photographed at 14 and 23 days post infection (DPI). These plants were infected
using bombardment by cloned viral DNA sequences. The CaLCuV specimens were photographed 11 and 18 DPI. These plants were infected using
an Agrobacteria carrying viral genomic DNA in T-DNA plasmids. All scale bars are approximate, but show that CaLCuV and the AMCV-Cam have
similar virulence.
Viral Replication
EACMV-UG3
ACMV-Cam
DNA Blot of the infected plants:
Leaf samples were collected 29
DPI and the DNA extracted using
a CTAB, phenol:chloroform
extraction. The ACMV-Cam
samples were digested with
XbahI, and the EACMV-UG3
samples were digested with NotI.
Lanes 1-2 show the ACMV-Cam
samples hybridized with
radiolabeled viral A component
DNA probes. Lane 1 shows the
single-stranded viral DNA and
the double stranded replication
intermediates in an infected
plant. Lane 2 shows the
absence of any viral DNA in a
non infected plant. Lanes 3-4
show the EACMV-UG3 samples
hybridized with radiolabeled viral
A component DNA probes. Lane
1 shows the absence of viral
DNA in the infected plants, and
lane 4 shows the absence of
viral DNA in the non infected
plants.
4
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Curling, Stunting and Chlorosis: The plants were scored daily for symptoms of
curling, stunting and chlorosis independently by visual inspection. Zero
represents no curling or chlorosis, 1 is slight curling and mosaic chlorosis, 2 is
moderate curling and chlorosis of veins, 3 is heavy curling and chlorosis between
veins, and 4 is severe curling and chlorosis of the entire leaf.
Summary and Conclusions
Relative Viral DNA Load
(EACMV-UG3)
1. All three viruses infect and replicate in N. benthamiana
2. CaLCuV and ACMV-Cam cause severe symptoms
characterized by chlorosis and stunting.
3. EACMV-UG3 causes very mild symptoms and replicates
poorly.
4. Viral DNA loads for EACMV-UG3 in infected plants are much
lower than the levels of ACMV-Cam or CaLCuV DNA.
Relative Viral DNA Load
(CaLCuV)
160000
160000
140000
140000
120000
120000
dsDNA100000
100000
80000
60000
ssDNA-
60000
40000
40000
20000
20000
0
A1
I
M
I
M
Acknowledgements
80000
B1
B3
C1
C4
0
M3
M4
C1
C2
C3
C4
Relative Viral DNA Loads: This is a quantification of the 29 DPI Uganda and 20 DPI CaLCuV dot blots above. The blue bars correspond to the mock infected plants, while
the green bars correspond to the infected plants. This shows the low amount of viral DNA in the EACMV-UG3 infected plants and high amounts in the CaLCuV infected
plants. The blots were probed using a radiolabeled A component of their virus. The image was quantified with ImageQuant.
Thanks to the Dr. Sue Carson at the North Carolina
State University Plant Biology Department and to
the National Science Foundation’s Research
Experience for Undergraduates Grant