SEGS-1 a cassava genomic sequence increases the severity of African cassava mosaic virus infection in Arabidopsis thaliana

Abstract

Cassava is a major crop in Sub-Saharan Africa, where it is grown primarily by smallholder farmers. Cassava production is constrained by Cassava mosaic disease (CMD), which is caused by a complex of cassava mosaic begomoviruses (CMBs). A previous study showed that SEGS-1 (sequences enhancing geminivirus symptoms), which occurs in the cassava genome and as episomes during viral infection, enhances CMD symptoms and breaks resistance in cassava. We report here that SEGS-1 also increases viral disease severity in Arabidopsis thaliana plants that are co-inoculated with African cassava mosaic virus (ACMV) and SEGS-1 sequences. Viral disease was also enhanced in Arabidopsis plants carrying a SEGS-1 transgene when inoculated with ACMV alone. Unlike cassava, no SEGS-1 episomal DNA was detected in the transgenic Arabidopsis plants during ACMV infection. Studies using Nicotiana tabacum suspension cells showed that co-transfection of SEGS-1 sequences with an ACMV replicon increases viral DNA accumulation in the absence of viral movement. Together, these results demonstrated that SEGS-1 can function in a heterologous host to increase disease severity. Moreover, SEGS-1 is active in a host genomic context, indicating that SEGS-1 episomes are not required for disease enhancement.

Keywords: ACMV; Arabidopsis thaliana; SEGS-1; begomovirus; cassava.

Figure 1

SEGS-1 enhances ACMV symptoms in Arabidopsis Sei-0. (A) SEGS-1 clones used for infection studies. The clones include a dimer (S1-2.0), a 1.5-mer with 2 GC-rich regions (S1-1.5a), a 1.5-mer with 1 GC-rich region (S1-1.5b), and a monomer (S1-1.0) that is configured like the full-length copy of SEGS-1 in the cassava genome. The gray segments and an embedded GC-rich region represent sequences that are duplicated in a construct. (B) Symptom development in plants inoculated with ACMV alone, ACMV + S1-1.5a, and S1-1.5a alone, or mock (ACMV DNA-B only). (C) Symptoms at 24 dpi in plants co-inoculated with ACMV and S1-1.0, S1-1.5a, S1-1.5b, or S1-2.0. (D) Time course of average symptom scores (1- no symptoms, 5- severe symptoms) for plants inoculated with ACMV alone or co-inoculated with ACMV + SEGS-1 clone. Values represent the mean of 10 plants per treatment. Asterisks (*) indicate significant differences between ACMV alone and ACMV+SEGS-1 treatments (p < 0.05 in a Wilcoxon ranked sum test).

Figure 2

SEGS-1 increases ACMV DNA accumulation in Arabidopsis Sei-0. (A) End-point PCR using the ACMV divLF/ACMV divLR primer pair to amplify ACMV DNA-A in mock (M; lane 1), ACMV alone (lane 2), or ACMV co-inoculated with S1-1.0 (lane 3), S1-1.5a (lane 4), S1-1.5b (lane 5) or S1-2.0 (lane 6) at 24 dpi. A negative no template control and a cloned positive plasmid DNA control are indicated by –C and +C (lanes 7 and 8, respectively). (B) In situ hybridization of ACMV DNA-A in plants at 24 dpi with ACMV alone or co-inoculated with ACMV and the indicated SEGS-1 clone. The 415-bp, DIG-labeled, DNA-A-specific probe forms a black precipitate over virus-positive nuclei. The leaf sections correspond to regions with vascular bundles where ACMV localizes. Mock plants were inoculated with ACMV DNA-B alone and did not contain infected cells. (C) Statistical analyses of virus-positive nuclei counts ( Supplementary Table 1 ) from in situ hybridization images using two-tailed paired Student’s t-test. Values in bold indicate significant differences (P<0.05).

Figure 3

SEGS-1 enhances ACMV DNA-A accumulation in tobacco protoplasts. (A) DNA gel blot showing the accumulation of nascent double-stranded ACMV DNA-A in protoplasts from NT-1 suspension cells at 48 h post transfection. The transfections are mock (pUC119: empty vector control), DNA-A + pUC119 (lanes 4-6), DNA-A + S1-1.0 (lanes 7-9), DNA-A + S1-1.5a (lanes 10-12), DNA-A + S1-1.5a (lanes 13-15) and DNA-A + S1-2.0 (lanes 16-18). The blot was hybridized to a 947-bp ACMV DNA-A fragment labeled with ³²P and visualized by phosphor imaging. (B) ³²P pixels were quantified using GelQuant software. Values represent the mean of 3 replicates/treatment. Bars correspond to ± 2 standard errors from the mean. Asterisks (*) indicate significant differences between the ACMV + empty vector treatment and an ACMV + SEGS-1 treatment (p < 0.05 in a two-tailed Student’s t test).

Figure 4

A SEGS-1 transgene enhances ACMV infection in Arabidopsis Sei-0 plants. (A) Time course (10, 17 and 24 dpi) of symptom development after inoculation with ACMV DNA-A + DNA-B or ACMV B alone (mock) in wild-type plants and in transgenic plants carrying a monomeric SEGS-1 transgene in the forward (S1-1.0F) or a reverse (S1-1.0R) orientation. (B) Time course of average symptom scores for wild-type Sei-0, S1-1.0F and S1-1.0R plants inoculated with ACMV at 10, 17, 24 and 31 dpi. Values represent the mean of 10 plants per treatment. Asterisks (*) indicate significant differences between ACMV alone and ACMV+SEGS-1 treatments (p < 0.05 in a Wilcoxon ranked sum test). (C) ACMV DNA-A copy number/ng total DNA in infected wild-type, S1-1.0F and S1-1.0R plants. The values represent the mean of 4 plants/treatment. Bars correspond to ± 2 standard errors from the mean. The ACMV DNA-A copy numbers in S1-1.0F and S1-1.0R plants were higher than in wild-type plants at 17 and 24 dpi, but no significant differences between the means were detected between the treatments by two-tailed Student’s t tests. The bars represent ± 2 standard errors from the mean. (D) Ratios of ACMV DNA-A mean copy numbers in S1-1.0F or S1-1.0R plants relative to wild-type plants. The dotted line represents the copy number in wild-type plants set to 1. (E) In situ hybridization of ACMV DNA-A in wild-type Sei-0, S1-1.0F and S1-1.0R plants at 10, 17 or 24 dpi with ACMV. The 415-bp DIG-labeled, DNA-A-specific probe forms a black precipitate over virus-positive nuclei. The leaf sections correspond to regions with vascular bundles where ACMV localizes. Mock plants were inoculated with ACMV DNA-B and did not contain infected cells. (F) Statistical analyses of virus-positive nuclei counts ( Supplementary Table 2 ) from in situ hybridization images using two-tailed paired Student’s t-test. Values in bold indicate significant differences (P<0.05). (G) SEGS-1 episome analysis. The divergent primer pair 1-4F/1-2R ( Table 1 ) was used to detect SEGS-1 episomes after RCA of total DNA. The top gel shows no PCR products amplifying across the SEGS-1 episome junction in ACMV-inoculated wild-type (wt; lane 2), S1-1.0F (lanes 3 and 4), and S1-1.0R (lanes 5 and 6) plants. C- is the water only negative PCR control (lane 7). C+ is the positive PCR control using SEGS-1 plasmid DNA as template that amplified in parallel with the Arabidopsis samples (lane 8). The bottom gel shows end-point PCR analysis using the CMAFor4/CMARev4 primer pair to amplify ACMV DNA-A in the same Arabidopsis DNA samples. Mock (lane 1) is DNA from an Arabidopsis plant inoculated with ACMV DNA-B only. In lanes 9 and 10, DNA samples from uninfected and ACMV-infected cassava plants were analyzed in parallel using the same protocol as the Arabidopsis episome assays. SEGS-1 episomes were detected in the DNA sample analyzed in lane 10 but not in the sample in lane 9.