Symptom Comparison

Alex Rajewski1, Mary Beth Dallas2, and Linda Hanley-Bowdoin2

1Biochemistry, Cell & Molecular Biology Program, Drake University, Des Moines IA 50311 2Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh NC 27606

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.

Viral Replication

AbstractThe 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.

These studies examine Cabbage leaf curl virus (CaLCuV), African cassava mosaic virus-Cameroon (ACMV-Cam) and East African cassava mosaic virus-Uganda (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.

Summary and Conclusions1. All three viruses infect and replicate in N. benthamiana2. 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.

Acknowledgements

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

Comparison of the Symptoms Caused by Three Geminiviruses in a Common Host

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.

ACMV-Cam EACMV-UG3 (CaLCuV)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 240

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Chlorosis

Stunting and Curling

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.

ACMV-Cam

EACMV-UG3

CaLCuV

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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.

Relative Viral DNA Load(EACMV-UG3)

Relative Viral DNA Load(CaLCuV)

A1 B1 B3 C1 C40

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