Catherine D Aimone, Leandro De León, Mary M Dallas, Joseph Ndunguru, José T Ascencio-Ibáñez , Linda Hanley-Bowdoin.

Journal of Virology; 2021 Oct 13;95(21):e0043221.

 doi: 10.1128/JVI.00432-21.

Abstract

Cassava mosaic disease (CMD), which is caused by single-stranded DNA begomoviruses, severely limits cassava production across Africa. A previous study showed that CMD symptom severity and viral DNA accumulation increase in cassava in the presence of a DNA sequence designated SEGS-2 (sequence enhancing geminivirus symptoms). We report here that when SEGS-2 is coinoculated with African cassava mosaic virus (ACMV) onto Arabidopsis thaliana, viral symptoms increase. Transgenic Arabidopsis with an integrated copy of SEGS-2 inoculated with ACMV also display increased symptom severity and viral DNA levels. Moreover, SEGS-2 enables Cabbage leaf curl virus (CaLCuV) to infect a geminivirus-resistant Arabidopsis thaliana accession. Although SEGS-2 is related to cassava genomic sequences, an earlier study showed that it occurs as episomes and is packaged into virions in CMD-infected cassava and viruliferous whiteflies. We identified SEGS-2 episomes in SEGS-2 transgenic Arabidopsis. The episomes occur as both double-stranded and single-stranded DNA, with the single-stranded form packaged into virions. In addition, SEGS-2 episomes replicate in tobacco protoplasts in the presence, but not the absence, of ACMV DNA-A. SEGS-2 episomes contain a SEGS-2 derived promoter and an open reading frame with the potential to encode a 75-amino acid protein. An ATG mutation at the beginning of the SEGS-2 coding region does not enhance ACMV infection in A. thaliana. Together, the results established that SEGS-2 is a new type of begomovirus satellite that enhances viral disease through the action of an SEGS-2-encoded protein that may also be encoded by the cassava genome. 

IMPORTANCE Cassava is an important root crop in the developing world and a food and income crop for more than 300 million African farmers. Cassava is rising in global importance and trade as the demands for biofuels and commercial starch increase. More than half of the world's cassava is produced in Africa, where it is primarily grown by smallholder farmers, many of whom are from the poorest villages. Although cassava can grow under high temperature, drought, and poor soil conditions, its production is severely limited by viral diseases. Cassava mosaic disease (CMD) is one of the most important viral diseases of cassava and can cause up to 100% yield losses. We provide evidence that SEGS-2, which was originally isolated from cassava crops displaying severe and atypical CMD symptoms in Tanzanian fields, is a novel begomovirus satellite that can compromise the development of durable CMD resistance.

See: https://pubmed.ncbi.nlm.nih.gov/34406866/

FIG. 3: SEGS-2 episomes are packaged into virions. (A) The divergent primer pair 2-4F/6R (indicated by small arrowheads on the episome and linear model) was used to detect episomal copies after RCA of total DNA. The large arrows indicate the SEGS-2 episome junction site. (B) PCR products from mock (M) and ACMV-inoculated (I) wild-type (WT; lanes 1 and 2) and transgenic (T-S2-1.0R; lanes 3 and 4) Sei-0 leaves. The bottom gel, which is enhanced 10-fold, shows episomes in both mock-infected and infected T-S2-1.0R leaves (lanes 3 and 4). (C) PCR of RCA products from mock (M) and ACMV-inoculated wild-type (WT; lanes 1 and 2) and WT+S2-1.0 (lanes 3 and 4) Sei-0 leaves. (D) PCR of RCA products from virion DNA of mock-inoculated (M, lane 1) and ACMV-infected (I, lane 2) T-S2-1.0F plants. The convergent primer pair PCNA2-F/R verified that there was no genomic DNA contamination of virion RCA template (bottom gel lanes 1 and 2). (E) PCR of RCA products of virion and total DNA treated with mung bean nuclease (+MB, lane 2) or not treated (−MB, lane 1). (F) Amplification of SEGS-2 episome in replication assays using protoplasts from N. tabacum suspension cells. Episomes were analyzed in protoplasts cotransfected with ACMV DNA-A + S2-1.0 (lanes 5 and 6), S2-1.5a (lanes 7 and 8), or S2-1.5b (lanes 9 and 10). ACMV DNA-A was transfected alone in lane 1. The SEGS-2 plasmids were transfected alone in lanes 2 (S2-1.0), 3 (S2-1.5a), and 4 (S2-1.5b). The replication assays were repeated three times. The positive control (C+) used S2-1.5b plasmid DNA as the template, and the negative control (C−) did not contain template DNA.