The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont.

• The arbuscular mycorrhizal symbiosis is arguably the most ecologically important eukaryotic symbiosis, yet it is poorly understood at the molecular level. To provide novel insights into the molecular basis of symbiosis-associated traits, we report the first genome-wide analysis of the transcriptome from Glomus intraradices DAOM 197198. • We generated a set of 25,906 nonredundant virtual transcripts (NRVTs) transcribed in germinated spores, extraradical mycelium and symbiotic roots using Sanger and 454 sequencing. NRVTs were used to construct an oligoarray for investigating gene expression. • We identified transcripts coding for the meiotic recombination machinery, as well as meiosis-specific proteins, suggesting that the lack of a known sexual cycle in G. intraradices is not a result of major deletions of genes essential for sexual reproduction and meiosis. Induced expression of genes encoding membrane transporters and small secreted proteins in intraradical mycelium, together with the lack of expression of hydrolytic enzymes acting on plant cell wall polysaccharides, are all features of G. intraradices that are shared with ectomycorrhizal symbionts and obligate biotrophic pathogens. • Our results illuminate the genetic basis of symbiosis-related traits of the most ancient lineage of plant biotrophs, advancing future research on these agriculturally and ecologically important symbionts.

Replication of the DNA A component of African cassava mosaic virus in a heterologous system.

The capacity for autonomous replication of the DNA A of African cassava mosaic virus (ACMV), a member of the bipartite geminiviruses infecting dicotyledonous plants, has been compared in host and non-host cells. A derivative of the ACMV DNA A was transfected into tobacco and maize protoplasts. Although ACMV is not able to infect maize, replication of the DNA A in maize protoplasts was observed to occur. The efficiency of replication was 10 to 20% of that seen in tobacco protoplasts. In both plant systems, replication was detected after the onset of cell division. ACMV replication in maize cells was compared to that of wheat dwarf virus and found to be 10 to 20% of that observed with the monocotyledon-specific virus. Insertion of 1165 bp of non-viral DNA into the ACMV DNA A prevented replication in maize but not in tobacco.

Cytosine methylation inhibits replication of African cassava mosaic virus by two distinct mechanisms.

Extrachromosomally replicating viral DNA is usually free of cytosine methylation and viral templates methylated in vitro are poor substrates when used in replication assays. We have investigated the mechanism of inhibition of viral replication by DNA methylation using as a model the DNA A of African cassava mosaic virus. We have constructed two component helper systems which allow for separation of the transcriptional inhibition of viral genes necessary for replication from replication inhibition due to altered interaction between the replication complex and methylated viral DNA. Our results suggest that methylation-mediated reduction of viral replication is due to both repression mechanisms and that this provides two independent selection pressures for the maintenance of methylation-free replicons in infected cells.

A parsley 4CL-1 promoter fragment specifies complex expression patterns in transgenic tobacco.

The 4CL-1 gene is one of two highly homologous parsley genes encoding 4-coumarate:coenzyme A ligase, a key enzyme of general phenylpropanoid metabolism. Expression of these genes is essential for the biosynthesis of both defense-related and developmentally required phenylpropanoid derivatives. We examined the developmental regulation of the 4CL-1 promoter by analyzing the expression of 4CL-1-beta-glucuronidase fusions in transgenic tobacco plants. A 597-base pair 4CL-1 promoter fragment specified histochemically detectable expression in a complex array of vegetative and floral tissues and cell types. The activity of a series of 5' deleted promoter fragments was analyzed in parsley protoplasts and transgenic tobacco plants. Deletions past -210 base pairs led to a drastic decline in beta-glucuronidase activity in protoplasts and loss of tissue-specific expression in transgenic tobacco. These results were put into the context of potential protein-DNA interactions by in vivo footprint analysis of the 4CL-1 promoter in parsley cells. Loss of promoter activity in parsley protoplasts and transgenic tobacco was correlated with the deletion or disruption of the distal portion of a large (100-base pair) footprinted region within the first 200 base pairs of the 4CL-1 promoter.

Exonic sequences are required for elicitor and light activation of a plant defense gene, but promoter sequences are sufficient for tissue specific expression.

The parsley 4CL-1 gene encodes 4-coumarate:CoA ligase, a key enzyme of general phenylpropanoid metabolism. As well as being transcriptionally activated by such stresses as pathogen infection, UV-irradiation, and wounding, expression of 4CL-1 is developmentally regulated. In this paper we present evidence that 4CL-1 cis-acting elements which control stress-induced and developmental expression are physically separated. The ability of a series of 4CL gene constructions to respond to elicitor and light in stably or transiently transformed parsley cells was tested. While inducible expression was observed from all templates in which the 4CL-1 structural gene was fused to the 4CL-1 promoter, fusions of the promoter to the GUS reporter gene were completely unresponsive. The element(s) required for responsiveness appear to be exonic, since 4CL-1 introns and 3' flanking DNA had no effect on inducibility. Furthermore, this unconventional regulatory mode operates in transgenic tobacco plants, where we show that a 4CL-1 promoter fragment specifies correct cell-specific expression when fused to GUS yet is unresponsive to elicitor and light.