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.

Effects and effectiveness of two RNAi constructs for resistance to Pepper golden mosaic virus in Nicotiana benthamiana plants.

ToChLPV and PepGMV are Begomoviruses that have adapted to a wide host range and are able to cause major diseases in agronomic crops. We analyzed the efficacy of induced resistance to PepGMV in Nicotiana benthamiana plants with two constructs: one construct with homologous sequences derived from PepGMV, and the other construct with heterologous sequences derived from ToChLPV. Plants protected with the heterologous construct showed an efficacy to decrease the severity of symptoms of 45%, while plants protected with the homologous construct showed an efficacy of 80%. Plants protected with the heterologous construct showed a reduction of incidence of 42.86%, while the reduction of incidence in plants protected with the homologous construct was 57.15%. The efficacy to decrease viral load was 95.6% in plants protected with the heterologous construct, and 99.56% in plants protected with the homologous construct. We found, in both constructs, up-regulated key components of the RNAi pathway. This demonstrates that the efficacy of the constructs was due to the activation of the gene silencing mechanism, and is reflected in the decrease of viral genome copies, as well as in recovery phenotype. We present evidence that both constructs are functional and can efficiently induce transient resistance against PepGMV infections. This observation guarantees a further exploration as a strategy to control complex Begomovirus diseases in the field.

Virus-induced silencing of Comt, pAmt and Kas genes results in a reduction of capsaicinoid accumulation in chili pepper fruits.

Capsaicinoids are responsible for the pungent taste of chili pepper fruits of Capsicum species. Capsaicinoids are biosynthesized through both the phenylpropanoid and the branched-fatty acids pathways. Fragments of Comt (encoding a caffeic acid O-methyltransferase), pAmt (a putative aminotransferase), and Kas (a beta-keto-acyl-[acyl-carrier-protein] synthase) genes, that are differentially expressed in placenta tissue of pungent chili pepper, were individually inserted into a Pepper huasteco yellow veins virus (PHYVV)-derived vector to determine, by virus-induced gene silencing, irrespective of whether these genes are involved in the biosynthesis of capsaicinoids. Reduction of the respective mRNA levels as well as the presence of related siRNAs confirmed the silencing of these three genes. Morphological alterations were evident in plants inoculated with PHYVV::Comt and PHYVV::Kas constructs; however, plants inoculated with PHYVV::pAmt showed no evident alterations. On the other hand, fruit setting was normal in all cases. Biochemical analysis of placenta tissues showed that, indeed, independent silencing of all three genes led to a dramatic reduction in capsaicinoid content in the fruits demonstrating the participation of these genes in capsaicinoid biosynthesis. Using this approach it was possible to generate non-pungent chili peppers at high efficiency.