Mutation of the conserved late element in geminivirus CP promoters abolishes Arabidopsis TCP24 transcription factor binding and decreases H3K27me3 levels on viral chromatin.

In geminiviruses belonging to the genus Begomovirus, coat protein (CP) expression depends on viral AL2 protein, which derepresses and activates the CP promoter through sequence elements that lie within the viral intergenic region (IR). However, AL2 does not exhibit sequence-specific DNA binding activity but is instead directed to responsive promoters through interactions with host factors, most likely transcriptional activators and/or repressors. In this study, we describe a repressive plant-specific transcription factor, Arabidopsis thaliana TCP24 (AtTCP24), that interacts with AL2 and recognizes a class II TCP binding site in the CP promoter (GTGGTCCC). This motif corresponds to the previously identified conserved late element (CLE). We also report that histone 3 lysine 27 trimethylation (H3K27me3), an epigenetic mark associated with facultative repression, is enriched over the viral IR. H3K27me3 is deposited by Polycomb Repressive Complex 2 (PRC2), a critical regulator of gene expression and development in plants and animals. Remarkably, mutation of the TCP24 binding site (the CLE) in tomato golden mosaic virus (TGMV) and cabbage leaf curl virus (CaLCuV) CP promoters greatly diminishes H3K27me3 levels on viral chromatin and causes a dramatic delay and attenuation of disease symptoms in infected Arabidopsis and Nicotiana benthamiana plants. Symptom remission is accompanied by decreased viral DNA levels in systemically infected tissue. Nevertheless, in transient replication assays CLE mutation delays but does not limit the accumulation of viral double-stranded DNA, although single-stranded DNA and CP mRNA levels are decreased. These findings suggest that TCP24 binding to the CLE leads to CP promoter repression and H3K27me3 deposition, while TCP24-AL2 interaction may recruit AL2 to derepress and activate the promoter. Thus, a repressive host transcription factor may be repurposed to target a viral factor essential for promoter activity. The presence of the CLE in many begomoviruses suggests a common scheme for late promoter regulation.

Acylsugars protect Nicotiana benthamiana against insect herbivory and desiccation.

KEY MESSAGE: Nicotiana benthamiana acylsugar acyltransferase (ASAT) is required for protection against desiccation and insect herbivory. Knockout mutations provide a new resource for investigation of plant-aphid and plant-whitefly interactions. Nicotiana benthamiana is used extensively as a transient expression platform for functional analysis of genes from other species. Acylsugars, which are produced in the trichomes, are a hypothesized cause of the relatively high insect resistance that is observed in N. benthamiana. We characterized the N. benthamiana acylsugar profile, bioinformatically identified two acylsugar acyltransferase genes, ASAT1 and ASAT2, and used CRISPR/Cas9 mutagenesis to produce acylsugar-deficient plants for investigation of insect resistance and foliar water loss. Whereas asat1 mutations reduced accumulation, asat2 mutations caused almost complete depletion of foliar acylsucroses. Three hemipteran and three lepidopteran herbivores survived, gained weight, and/or reproduced significantly better on asat2 mutants than on wildtype N. benthamiana. Both asat1 and asat2 mutations reduced the water content and increased leaf temperature. Our results demonstrate the specific function of two ASAT proteins in N. benthamiana acylsugar biosynthesis, insect resistance, and desiccation tolerance. The improved growth of aphids and whiteflies on asat2 mutants will facilitate the use of N. benthamiana as a transient expression platform for the functional analysis of insect effectors and resistance genes from other plant species. Similarly, the absence of acylsugars in asat2 mutants will enable analysis of acylsugar biosynthesis genes from other Solanaceae by transient expression.

Medios de cultivo líquidos: un avance para la micropropagación comercial de zábila (Aloe barbadensis Mill.) / Liquid medium culture: an approach for the commercial micropropagation of aloe (Aloe barbadensis Mill.)

La micropropagación es una alternativa para la producción comercial de plantas de zábila (Aloe barbadensis Mill.) limitada por los altos costos de producción. Con el objetivo de prescindir de los agentes gelificantes, reduciendo costos, se comparó el medio de cultivo líquido con el medio de cultivo gelificado en las diferentes etapas de micropropagación de la zábila. En la etapa de establecimiento se observó mayor porcentaje de explantes contaminados en el medio de cultivo líquido estático (25.55 %) que en el medio gelificado (11.11 %); y aunque el resto de los explantes se establecieron independientemente de la condición del medio de cultivo, en el medio líquido alcanzaron mayor altura (3.81 cm) que en el medio gelificado (3.03 cm). En la etapa de multiplicación, la altura de los explantes (entre 4.43 y 6.01 cm) fue superior en los recipientes de inmersión temporal automatizado (RITA®) en comparación con el medio gelificado (entre 3.24 y 3.42 cm); sin diferencias significativas entre el número de brotes/explante. Todos los brotes enraizaron a los 30 días independientemente del medio de cultivo empleado (líquido estático y gelificado), sin observar variaciones en la altura del brote y, número y longitud de las raíces. El empleo de los medios de cultivo líquidos y la implementación de los sistemas de inmersión temporal tipo RITA® permiten reducir los costos de producción al prescindir de los agentes gelificantes, lo que representa un avance para la micropropagación comercial de zábila.

Micropropagation is considered a successful alternative for aloe (Aloe barbadensis Mill.) plant production. However, it has limited use due to the high production cost. Liquid media were compared to agar-gelled medium during all micropropagation stages of aloe to reduce the cost for gelling agent used. In the establishment stage, there was a higher percentage of contaminated explants in static liquid medium (25.55%) than those cultured in agar-gelled medium (11.11%), although all the explants were established independently of the culture medium used, higher height (3.81 cm) was observed in liquid medium than those growing in agar-gelled medium (3.03 cm). In the multiplication stage, explant height was higher in the recipients used for automated temporary immersion system (RITA®) (4.43 - 6.01 cm) than those cultured in agar-gelled medium (3.24 - 3.42 cm), there was no significant difference for number of shoots/explant. All shoots had roots at 30 days independently of used culture media (static liquid or agar-gelled media). Shoot height, number and root length had similar values in both culture media. The implementation of liquid media and automated temporary Immersion system RITA® may allow to reduce production costs of gelling agent used, it represents an approach for the commercial micropropagation of aloe.