Reactions of Nicotiana species to inoculation with monopartite and bipartite begomoviruses

Abstract

Background: Some Nicotiana species are widely used as experimental hosts for plant viruses. Nicotiana species differ in ploidy levels, chromosome numbers and have diverse geographical origins. Thus, these species are useful model systems to investigate virus-host interactions, co-evolution of pathogens and hosts and the effects of ploidy level on virus resistance/susceptibility.

Results: Here we have studied the responses of seven Nicotiana species to inoculation with Cotton leaf curl Multan virus (CLCuMV), a monopartite begomovirus, and Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, both from the Indian subcontinent. All Nicotiana species supported the replication of both begomoviruses in inoculated leaves. However, only three Nicotiana species, namely N. benthamiana, N. tabacum and N. sylvestris showed symptoms when inoculated with ToLCNDV, while N. benthamiana was the only species that developed leaf curl symptoms when inoculated with CLCuMV. CLCuMV accumulated to detectable levels in N. tabacum, but plants remained asymptomatic. A previously identified mutation of RNA dependent RNA polymerase 1 was shown to be present only in N. benthamiana. The finding is in line with earlier results showing that the susceptibility of this species to a diverse range of plant viruses correlates with a defective RNA silencing-mediated host defense.

Conclusions: The results presented show that individual Nicotiana species respond differently to inoculation with begomoviruses. The inability of begomoviruses to systemically infect several Nicotiana species is likely due to inhibition of virus movement, rather than replication, and thus provides a novel model to study virus-host interactions in resistant/susceptible hosts.

Figure 1

Photographs of Nicotiana species at 25 days after inoculation with CLCuMV. Shown are photographs of non-inoculated N. benthamiana (A), N. sylvestris (C), N. nudicaulis (E), N. obtusifolia (G), N. tabacum (I), N. suaveolens (K) and N. repanda (M) plants and photographs of N. benthamiana (B), N. sylvestris (D), N. nudicaulis (F), N. obtusifolia (H), N. tabacum (J), N. suaveolens (L) and N. repanda (N) plants inoculated with CLCuMV.

Figure 2

PCR-mediated detection of CLCuMV in inoculated Nicotiana plants. The ethidium bromide-stained agarose gel was photographed under UV illumination. The samples loaded on the gel resulted from PCR reactions with primer pair PK3AV2F/PK3AV2R and DNA extracted from the leaves of plants (as indicated above each well) inoculated with CLCuMV. The leaves sampled were developing at the time of, or developed after, inoculation and were sampled at 25 dpi. The presence of a 311 bp band indicates the systemic movement of CLCuMV from the site of inoculation. The sample in lane C resulted from PCR amplification with DNA extracted from a healthy N. benthamiana plant. A DNA size marker was electrophoresed in lane M. The sizes (bp) of selected marker bands are indicated on the left.

Figure 3

Southern blot detection of CLCuMV in inoculated Nicotiana plants. The DNA samples loaded on the gel were extracted from leaves of plants (as indicated above each well) inoculated with CLCuMV. The leaves sampled were developing at the time of, or developed after, inoculation and were sampled at 25 dpi. An approximately equal amount of DNA (10 μg) was loaded in each case. The blot was probed with a DIG-labeled CLCuMV fragment. The positions of replicative forms of viral DNA are indicated as open circular (oc), linear (lin), super coiled (sc) and single stranded (ss). A photograph of the genomic DNA bands on the ethidium bromide stained agarose gel to confirm equal loading is shown at the base.

Figure 4

PCR-mediated amplification of the full-length genome of CLCuMV from inoculated Nicotiana plants. The ethidium bromide-stained agarose gel was photographed under UV illumination. The samples loaded on the gel resulted from PCR reactions with primer pair BegomoF/BegomoR and DNA extracted from the leaves of plants inoculated with CLCuMV (as indicated above each well). The samples in lanes C1 and C2 resulted from PCR reactions with DNA extracted from a healthy N. benthamiana plant and the plasmid containing the full-length genome of CLCuMV, respectively. Possible sub-genomic virus fragments are highlighted with white arrows. A DNA size marker was electrophoresed in lanes M. The sizes (bp) of selected marker bands are indicated on the left.

Figure 5

Southern blot detection of CLCuMV in the inoculated tissues of Nicotiana plants. The DNA samples loaded on the gel were extracted from leaves of plants (as indicated above each well) inoculated with CLCuMV. The leaves sampled were those inoculated with CLCuMV and were sampled at 25 dpi. An approximately equal amount of DNA (10 μg) was loaded in each case. The blot was probed with DIG-labeled CLCuMV fragment. The positions of replicative forms of viral DNA are indicated as open circular (oc), linear (lin), super coiled (sc) and single stranded (ss). A photograph of the genomic DNA bands on the ethidium bromide stained agarose gel to confirm equal loading is shown at the base.

Figure 6

Symptoms exhibited by three Nicotiana species infected with ToLCNDV. Photographs of healthy N. benthamiana plant (A), N. sylvestris (C) and N. tabacum (E) as well as N. benthamiana plant (B), N. sylvestris (D) and N. tabacum (F) plants infected with ToLCNDV. Pictures were taken at 21 dpi.

Figure 7

PCR-mediated detection of ToLCNDV in inoculated Nicotiana plants. The ethidium bromide-stained agarose gel was photographed under UV illumination. The samples loaded on the gel resulted from PCR reactions with primer pair ToLCNDV2F/ToLCNDV2R and DNA extracted from the leaves of plants (as indicated above each well) inoculated with ToLCNDV. The leaves sampled were developing at the time of, or developed after, inoculation and were sampled at 25 dpi. The presence of a 352 bp band indicates the systemic movement of ToLCNDV from the site of inoculation. The sample in lane C resulted from PCR amplification with DNA extracted from a healthy N. benthamiana plant. A DNA size marker was electrophoresed in lane M. The sizes (bp) of selected marker bands are indicated on the left.

Figure 8

Southern blot detection of ToLCNDV in inoculated Nicotiana plants. The DNA samples loaded on the gel were extracted from leaves of plants (as indicated above each well) inoculated with ToLCNDV. The leaves sampled were developing at the time of, or developed after, inoculation and were sampled at 25 dpi. An approximately equal amount of DNA (10 μg) was loaded in each case. The blot was probed with DIG-labeled ToLCNDV fragment. The positions of replicative forms of viral DNA are indicated as open circular (oc), linear (lin) and single stranded (ss). A photograph of the genomic DNA bands on the ethidium bromide stained agarose gel to confirm equal loading is shown at the base.

Figure 9

Identification of mutated RDR1 in Nicotiana species. Shown is an ethidium bromide-stained agarose gel photographed under UV illumination. Samples loaded resulted from PCR reactions using primer pair RDRf/RDRr and DNA extracted from leaves of the Nicotiana species. The sample in lane C resulted from a PCR reaction with primers RDRf/RDRr but lacking template DNA. A DNA size marker was electrophoresed in lane M. The sizes (bp) of selected marker bands are indicated on the left.