A transposon surveillance mechanism that safeguards plant male fertility during stress.

Although plants are able to withstand a range of environmental conditions, spikes in ambient temperature can impact plant fertility causing reductions in seed yield and notable economic losses1,2. Therefore, understanding the precise molecular mechanisms that underpin plant fertility under environmental constraints is critical to safeguarding future food production3. Here, we identified two Argonaute-like proteins whose activities are required to sustain male fertility in maize plants under high temperatures. We found that MALE-ASSOCIATED ARGONAUTE-1 and -2 associate with temperature-induced phased secondary small RNAs in pre-meiotic anthers and are essential to controlling the activity of retrotransposons in male meiocyte initials. Biochemical and structural analyses revealed how male-associated Argonaute activity and its interaction with retrotransposon RNA targets is modulated through the dynamic phosphorylation of a set of highly conserved, surface-located serine residues. Our results demonstrate that an Argonaute-dependent, RNA-guided surveillance mechanism is critical in plants to sustain male fertility under environmentally constrained conditions, by controlling the mutagenic activity of transposons in male germ cells.

DNA methylation of retrotransposons, DNA transposons and genes in sugar beet (Beta vulgaris L.).

The methylation of cytosines shapes the epigenetic landscape of plant genomes, coordinates transgenerational epigenetic inheritance, represses the activity of transposable elements (TEs), affects gene expression and, hence, can influence the phenotype. Sugar beet (Beta vulgaris ssp. vulgaris), an important crop that accounts for 30% of worldwide sugar needs, has a relatively small genome size (758 Mbp) consisting of approximately 485 Mbp repetitive DNA (64%), in particular satellite DNA, retrotransposons and DNA transposons. Genome-wide cytosine methylation in the sugar beet genome was studied in leaves and leaf-derived callus with a focus on repetitive sequences, including retrotransposons and DNA transposons, the major groups of repetitive DNA sequences, and compared with gene methylation. Genes showed a specific methylation pattern for CG, CHG (H = A, C, and T) and CHH sites, whereas the TE pattern differed, depending on the TE class (class 1, retrotransposons and class 2, DNA transposons). Along genes and TEs, CG and CHG methylation was higher than that of adjacent genomic regions. In contrast to the relatively low CHH methylation in retrotransposons and genes, the level of CHH methylation in DNA transposons was strongly increased, pointing to a functional role of asymmetric methylation in DNA transposon silencing. Comparison of genome-wide DNA methylation between sugar beet leaves and callus revealed a differential methylation upon tissue culture. Potential epialleles were hypomethylated (lower methylation) at CG and CHG sites in retrotransposons and genes and hypermethylated (higher methylation) at CHH sites in DNA transposons of callus when compared with leaves.

Small RNA activity and function in angiosperm gametophytes.

Small non-coding RNAs are key post-transcriptional and transcriptional regulators of plant gene expression in angiosperm sporophytes. In recent years, gametophytic small RNAs have also been investigated, predominantly in Arabidopsis male gametophytes, revealing features in common with the sporophyte as well as some surprising differences. Transcriptomic and deep-sequencing studies confirm that multiple small RNA pathways operate in male gametophytes, with over 100 miRNAs detected throughout development. Trans-acting siRNA pathways that are associated with novel phased transcripts in pollen, and the nat-siRNA pathway have important roles in pollen maturation and gamete function. Moreover, a role for siRNA-triggered silencing of transposable elements in male and female germ cells has been established, a feature in common with the role of piRNAs in animal germlines. Current evidence supports an integral role for small RNAs in angiosperm gametophyte development and it can be anticipated that novel small RNAs with significant roles in germline development and genome integrity await discovery.

Small RNAs in angiosperm gametophytes: from epigenetics to gamete development.

The established role of various small RNA pathways in the epigenetic regulation of gene expression in the dipolid sporophytic generation of flowering plants contrasts sharply with the lack of knowledge of their role in haploid gametophyte generation. Several recent studies now uncover the operation of multiple small RNA pathways in male and female gametophytes and their essential roles in genome integrity, cell specification, and, most recently, sperm cell function, as described in the May 15, 2010, issue of Genes & Development by Ron and colleagues (pp. 1010-1021).

Phenotypic mutants of the intracellular actinomycete Rhodococcus equi created by in vivo Himar1 transposon mutagenesis.

Rhodococcus equi is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses. An effective live attenuated vaccine would be extremely useful in the prevention of R. equi disease in horses. Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a Himar1 minitransposon delivered by a conditionally replicating plasmid for construction of R. equi mutants. We show that Himar1 transposition in R. equi is random and needs no apparent consensus sequence beyond the required TA dinucleotide. The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes. One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of R. equi. We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis. This mutant cannot grow in minimal medium in the absence of riboflavin supplementation. Experimental murine infection studies showed that, in contrast to wild-type R. equi, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo. The mutagenesis methodology we have developed will allow the characterization of R. equi virulence mechanisms and the creation of other attenuated strains with vaccine potential.

Tissue-specific effects of maize bronze gene promoter mutations induced by Ds1 insertion and excision.

Bz-wm is an allele of the Bz locus of maize isolated by McClintock (1962) as a derivative of bz-m2. It contains a Ds1 insertion 63 bp upstream of the start of transcription and a 3 bp insertion in the coding region at the site of the Ac element that was present in bz-m2. Bz-wm produces, in the aleurone layer of the endosperm, low amounts (approximately 1% of wild-type) of a Bz-gene encoded UDP-glucose: flavoid 3-0-glucosyltransferase (UFGT) polypeptide with altered thermal stability. Three phenotypically wild-type derivatives, Bz' (wm)-1, Bz' (wm)-2 and Bz' (wm)-3, were isolated in the presence of Ac and shown to have excised the Ds1 element but not fully restored UFGT activity in endosperm assays. In the studies reported here, we have further analyzed these Bz' derivatives of Bz-wm by determining the DNA sequences left behind on Ds1 excision, and by measuring the amount of UFGT activity and/or Bz mRNA conditioned by Bz-wm and the Bz' derivatives in different tissues. The data indicate that tissue-specific differences in expression of the Bz gene have been produced in alleles with mutations caused by transposable elements Ac and Ds. These mutations may affect either the amount of Bz transcription or the stability of the UFGT polypeptide. The sequence or spacing in the -63 region of the Bz promoter appears to be critical for maximum expression in aleurone and husk but not in pollen and pigmented seedling tissue.