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

UNLABELLED: 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. IMPORTANCE: Viral 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.

Genome-wide analysis of the beta-glucosidase gene family in maize (Zea mays L. var B73).

The hydrolysis of beta-D: -glucosidic bonds which is required for the liberation of many physiologically important compounds is catalyzed by the enzyme beta-glucosidase (BGLU, EC 3.2.1.21). BGLUs are implicated in several processes in plants, such as the timely response to biotic and abiotic stresses through activation of phytohormones and defense compounds. We identified 26 BGLU isozymes in the genome of the maize inbred B73 and propose a standardized nomenclature for all Zea mays BGLU paralogs (Zmbglu1-Zmbglu26). We characterized their intron-exon structure, protein features, phylogenetic relationships, and measured their expression and activity in various tissues under different environmental conditions. Sequence alignments revealed some characteristic motifs (conserved amino acids) and specific differences among different isozymes. Analysis of putative signal peptides suggested that some BGLUs are plastidic, whereas others are mitochondrial, cytosolic, vacuolar or secreted. Microarray and RT-PCR analysis showed that each member of the Zmbglu family had a characteristic expression pattern with regard to tissue specificity and response to different abiotic conditions. The source of variance for gene expression was highest for the type of organ analyzed (tissue variance) than for the growth conditions (environmental variance) or genotype (genetic variance). Analysis of promoter sequences revealed that each Zmbglu paralog possesses a distinct set of cis elements and transcription factor binding sites. Since there are no two Zmbglu paralogs that have identical molecular properties, we conclude that gene subfunctionalization in maize occurs much more rapidly than gene duplication.