Two Populations of Viral Minichromosomes Are Present in a Geminivirus-Infected Plant Showing Symptom Remission (Recovery)

Abstract

Geminiviruses are important plant pathogens characterized by circular, single-stranded DNA (ssDNA) genomes. However, in the nuclei of infected cells, viral double-stranded DNA (dsDNA) associates with host histones to form a minichromosome. In phloem-limited geminiviruses, the characterization of viral minichromosomes is hindered by the low concentration of recovered complexes due to the small number of infected cells. Nevertheless, geminiviruses are both inducers and targets of the host posttranscriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) machinery. We have previously characterized a "recovery" phenomenon observed in pepper plants infected with pepper golden mosaic virus (PepGMV) that is associated with a reduction of viral DNA and RNA levels, the presence of virus-related siRNAs, and an increase in the levels of viral DNA methylation. Initial micrococcal nuclease-based assays pinpointed the presence of different viral chromatin complexes in symptomatic and recovered tissues. Using the pepper-PepGMV system, we developed a methodology to obtain a viral minichromosome-enriched fraction that does not disturb the basic chromatin structural integrity, as evaluated by the detection of core histones. Using this procedure, we have further characterized two populations of viral minichromosomes in PepGMV-infected plants. After further purification using sucrose gradient sedimentation, we also observed that minichromosomes isolated from symptomatic tissue showed a relaxed conformation (based on their sedimentation rate), are associated with a chromatin activation marker (H3K4me3), and present a low level of DNA methylation. The minichromosome population obtained from recovered tissue, on the other hand, sedimented as a compact structure, is associated with a chromatin-repressive marker (H3K9me2), and presents a high level of DNA methylation. IMPORTANCEViral minichromosomes have been reported in several animal and plant models. However, in the case of geminiviruses, there has been some recent discussion about the importance of this structure and the significance of the epigenetic modifications that it can undergo during the infective cycle. Major problems in this type of studies are the low concentration of these complexes in an infected plant and the asynchronicity of infected cells along the process; therefore, the complexes isolated in a given moment usually represent a mixture of cells at different infection stages. The recovery process observed in PepGMV-infected plants and the isolation procedure described here provide two distinct populations of minichromosomes that will allow a more precise characterization of the modifications of viral DNA and its host proteins associated along the infective cycle. This structure could be also an interesting model to study several processes involving plant chromatin. G eminiviruses are important plant pathogens consisting of circular, single-stranded DNA (ssDNA) genomes ranging from 2.7 to 5.2 kb packed into 1 or 2 icosahedral twine-shaped part...

Mecanismos, aplicaciones y perspectivas del silenciamiento génico de virus en plantas

“…The most abundant size classes are 21 and 22 nt, and are made by DCL4 and DCL2, respectively (Deleris et al ., 2006) (Table 1). Virus-derived siRNAs mediate antiviral defense by inducing transcriptional and post-transcriptional gene silencing of viruses (Brosseau and Moffett, 2015; Ceniceros-Ojeda et al ., 2016). …”

“…Silencing is activated at the post-transcriptional level, resulting in the formation of siRNAs that guide targeting of viral RNA. Furthermore, virus-derived siRNAs in association of AGO and RDR proteins guide methylation of viral DNA, resulting in transcriptional gene silencing which prevents virus replication and movement (Raja et al ., 2008; Buchmann et al ., 2009; Ceniceros-Ojeda et al ., 2016). …”

“…Los ARNiv más abundantes miden 21 y 22 nt y son formados por DCL4 y DCL2, respectivamente (Deleris et al ., 2006) (Cuadro 1). Los ARNi derivados del virus regulan la defensa antiviral mediante la inducción del silenciamiento génico transcripcional y post-transcripcional de genes virales (Brosseau and Moffett, 2015; Ceniceros-Ojeda et al ., 2016). …”

“…Transcriptional gene silencing inhibits transcription, resulting in a reduction of mRNA, by methylating DNA in chromosomes, plasmids or viral minichromosomes (Raja et al ., 2008; Ceniceros-Ojeda et al ., 2016). Transcriptional gene silencing is directed by siRNAs guiding enzymatic DNA methylation (Ding and Voinnet, 2007).…”

“…El silenciamiento génico a nivel transcripcional inhibe la transcripción causando una reducción en la síntesis de ARN mensajero vía la metilación de ADN en cromosomas, plásmidos o minicromosomas virales (Raja et al ., 2008; Ceniceros-Ojeda et al ., 2016). El silenciamiento génico transcripcional es dirigido por los RNAi que guían la metilación enzimática del ADN (Ding and Voinnet, 2007).…”