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. 2011 May 19:8:238.
doi: 10.1186/1743-422X-8-238.

Comparison of phenotypes produced in response to transient expression of genes encoded by four distinct begomoviruses in Nicotiana benthamiana and their correlation with the levels of developmental miRNAs

Imran Amin  1 , Basavaprabhu L Patil, Rob W Briddon, Shahid Mansoor, Claude M Fauquet
Affiliations

Comparison of phenotypes produced in response to transient expression of genes encoded by four distinct begomoviruses in Nicotiana benthamiana and their correlation with the levels of developmental miRNAs

Imran Amin et al. Virol J. .

Abstract

Background: Whitefly-transmitted geminiviruses (begomoviruses) are a major limiting factor for the production of numerous dicotyledonous crops throughout the world. Begomoviruses differ in the number of components that make up their genomes and association with satellites, and yet they cause strikingly similar phenotypes, such as leaf curling, chlorosis and stunted plant growth. MicroRNAs (miRNAs) are small endogenous RNAs that regulate plant growth and development. The study described here was aimed at investigating the effects of each virus encoded gene on the levels of developmental miRNAs to identify common trends between distinct begomoviruses.

Results: All genes encoded by four distinct begomoviruses (African cassava mosaic virus [ACMV], Cabbage leaf curl virus [CbLCuV], Tomato yellow leaf curl virus [TYLCV] and Cotton leaf curl virus/Cotton leaf curl betasatellite [CLCuV/CLCuMB]) were expressed from a Potato virus X (PVX) vector in Nicotiana benthamiana. Changes in the levels of ten miRNAs in response to the virus genes were determined by northern blotting using specific miRNA probes. For the monopartite begomoviruses (TYLCV and CLCuMV) the V2 gene product was identified as the major symptom determinant while for bipartite begomoviruses (ACMV and CbLCuV) more than one gene appears to contribute to symptoms and this is reflected in changes in miRNA levels. The phenotype induced by expression of the βC1 gene of the betasatellite CLCuMB was the most distinct and consisted of leaf curling, vein swelling, thick green veins and enations and the pattern of changes in miRNA levels was the most distinct.

Conclusions: Our results have identified symptom determinants encoded by begomoviruses and show that developmental abnormalities caused by transient expression of begomovirus genes correlates with altered levels of developmental miRNAs. Additionally, all begomovirus genes were shown to modulate miRNA levels, the first time this has been shown to be the case.

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Figures

Figure 1
Figure 1
Symptoms exhibited by N. benthamiana plants infected with PVX expressing the rep genes of selected begomoviruses. Photographs of N. benthamiana plants, and close-up views of leaves, infected with PVX expressing the rep of ACMV (panels A and B), CLCuMV (panels C and D), CbLCuV (panels E and F). An N. benthamiana plant, and close-up view of a leaf, infected with the PVX vector (with no insert) are shown in panels G and H.
Figure 2
Figure 2
Effects of PVX-mediated expression of begomovirus genes on the levels of miR156, 159, 160, 164 and 165. The bar graphs show the average of two independent experiments (with standard error). The accumulation of each miRNA upon begomovirus gene expression from PVX was calculated relative to plants infected with PVX which was taken as 1. The genes used were those encoding the replication associated protein (Rep), the transcriptional activator protein (TrAP), the C2 protein (C2), the replication enhancer protein (REn), the (A)C4 protein [(A)C4], the coat protein (CP), the (A)V2 protein [(A)V2], the nuclear shuttle protein (NSP) and the movement protein (MP). Additionally the gene encoding the hypothetical protein C5 (C5*) of Cotton leaf curl Kokhran virus and the green fluorescence protein (GFP) were expressed from the PVX vector.
Figure 3
Figure 3
Effects of PVX-mediated expression of begomovirus genes on the levels of miR166, 167, 168, 169 and 170. The bar graphs show the average of two independent experiments (with standard error). The accumulation of each miRNA upon the expression of begomovirus genes from PVX was calculated relative to plants infected with PVX, which was taken as 1. The genes used were those encoding the replication associated protein (Rep), the transcriptional activator protein (TrAP), the C2 protein (C2), the replication enhancer protein (REn), the (A)C4 protein [(A)C4], the coat protein (CP), the (A)V2 protein [(A)V2], the nuclear shuttle protein (NSP) and the movement protein (MP). Additionally the gene encoding the hypothetical protein C5 (C5*) of Cotton leaf curl Kokhran virus and the green fluorescence protein (GFP) were expressed from the PVX vector.
Figure 4
Figure 4
Symptoms exhibited by N. benthamiana plants infected with PVX expressing the trap/c2 genes of selected begomoviruses. Photographs of N. benthamiana plants, and close up of leaves, infected with PVX expressing the trap/c2 of ACMV (panels A and B), CLCuMV (panels C and D), CbLCuV (panels E and F) and TYLCV (panels G and H).
Figure 5
Figure 5
A heat diagram summarising miRNA accumulation in response to PVX-mediated expression of selected begomovirus genes. The results shown are for N. benthamiana plants infected with PVX harbouring the trap/c2 genes (A), (a)c4 genes (B) and (a)v2 genes (C). miRNA accumulations were compared with PVX infection alone by taking that value as 1.
Figure 6
Figure 6
Symptoms exhibited by N. benthamiana plants infected with PVX expressing the ren genes of selected begomoviruses. Photographs of N. benthamiana plants, and close up of leaves, infected with PVX expressing ren of ACMV (panel A and B), CLCuMV (panel C and D), CbLCuV (panel E and F) and TYLCV (panel G and H).
Figure 7
Figure 7
Symptoms exhibited by N. benthamiana plants infected with PVX expressing the (a)c4 genes of selected begomoviruses. Photographs of N. benthamiana plants, and close up of leaves, infected with PVX expressing (a)c4 of ACMV (panels A and B), CLCuMV (panels C and D), CbLCuV (panels E and F) and TYLCV (panels G and H).
Figure 8
Figure 8
Symptoms exhibited by N. benthamiana plants infected with PVX expressing the cp genes of selected begomoviruses. Photographs of N. benthamiana plants, and close up of leaves, infected with PVX expressing cp of ACMV (panels A and B), CLCuMV (panel C and D), CbLCuV (panels E and F) and TYLCV (panels G and H).
Figure 9
Figure 9
Symptoms exhibited by N. benthamiana plants infected with PVX expressing the (a)v2 genes of selected begomoviruses. Photographs of N. benthamiana plants, and close up of leaves, infected with PVX expressing (a)v2 of ACMV (panels A and B), CLCuMV (panels C and D) and TYLCV (panels E and F).
Figure 10
Figure 10
Symptoms exhibited by N. benthamiana plants infected with PVX expressing the mp or nsp genes of selected begomoviruses. Photographs of N. benthamiana plants, and close-up views of leaves, infected with PVX expressing ACMV mp (panels A and B), ACMV nsp (panels C and D), CbLCuV mp (panels E and F) and CbLCuV nsp (panels G and H).
Figure 11
Figure 11
Symptoms exhibited by N. benthamiana plants infected with PVX expressing the c5 gene of CLCuKoV, the βc1 gene of CLCuMB and the gfp gene. Photographs of N. benthamiana plants, and close-up views of leaves, infected with PVX expressing CLCuKoV c5 (panels A and B), CLCuMB βc1 (panels C, D, E and F) and gfp (panels G and H). The photograph in panel H was taken under UV illumination to show GFP fluorescence.
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