Analysis of the promoters of four Pinus radiata male cone-specific genes in the model plant species Arabidopsis and tobacco

Full text is available to authenticated members of The University of Auckland only.Male sterility in Pinus radiata D. Don (P. radiata) was identified as a desirable trait and a targeted-cell-death approach (promoter::cytotoxic gene fusion) was chosen to achieve this goal. Four male cone-specific promoters were isolated from the genome of P. radiata, fused to the GUS reporter gene and the expression analysed in the heterologous host Arabidopsis thaliana (L.) Heynh (Arabidopsis). All four promoters PrCHS1, PrLPT2, PrMC2 and PrMALE1 directed expression in tapetum and microspores of Arabidopsis anthers however, three differing temporal expression patterns were observed. PrMC2 and PrMALE1 conferred identical anther expression patterns, possibly due to high sequence similarity (86%) within 276 bp upstream of the start codon. whole plant GUS expression patterns resulting from all four promoter::GUS fusion constructs showed a reocurring pattern in various parts of Arabidopsis plants, such as vascular bundles of roots, young leaves, sepals, filaments of stamens and in stems and pistils (especially the style). It was demonstrated that an almost identical expression pattern was induced by sequences of the backbone of the pCAMBIA binary vectors used (referred to as 'background' expression). In silico promoter analysis identified putative cis-acting elements within all isolated P. radiata promoters, potentially controlling tissue-specific expression in anthers/male cones. An assembly of three putative cis-acting elements was detected within the PrMALE1 promoter, less than 300 bp upstream of the translation start. However, deletion studies as well as gain-of-function experiments showed that a 16 bp motif of high homology to a known cis-acting element (anther-box) was not functional in the Arabidopsis genetic background. Likewise, deletion of a 10 bp motif (AAAAGAAGAA), which was found to be conserved in all four pine promoters, did not result in a specific loss of PrMALE1 promoter function in Arabidopsis. The deletion analysis showed that a 226 bp PrMALE1 promoter fragment encoded all sequence information necessary to confer temporal and spatial expression to the tapetum and developing microspores of Arabidopsis anthers. To demonstrate male sterility in tobacco, the full-length PrMALE1 promoter was transcriptionally fused to a stilbene synthase gene. Transgenic plants were produced via Agrobacterium-mediated leaf disk transformation. Fertility in all ten transgenic tobacco lines was severely affected and most lines showed 100% male sterility in pollen germination experiments. Anther development of male sterile tobacco plants was completely normal until microspores were release from tetrads. Released microspores and mature, ablated pollen grains differed in shape and Giemsa-staining color from wild-type pollen. Female fertility was not affected

Identification and Analysis of Expression of Novel MicroRNAs of Murine Gammaherpesvirus 68▿ †

Murine gammaherpesvirus 68 (MHV-68) is closely related to Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) and provides a small-animal model with which to study the pathogenesis of gammaherpesvirus (γHV) infections. To completely explore the potential of the MHV-68 system for the investigation of γHV microRNAs (miRNAs), it would be desirable to know the number and expression patterns of all miRNAs encoded by MHV-68. By deep sequencing of small RNAs, we systematically investigated the expression profiles of MHV-68 miRNAs in both lytically and persistently infected cells. In addition to the nine known MHV-68 miRNAs, we identified six novel MHV-68 miRNA genes and analyzed the expression levels of all MHV-68 miRNAs. Furthermore, we also characterized the cellular miRNA expression signatures in MHV-68-infected versus noninfected NIH 3T3 fibroblasts and in 12-O-tetradecanoyl-phorbol-13-acetate (TPA)-treated versus nontreated S11 cells. We found that mmu-mir-15b and mmu-mir-16 are highly upregulated upon MHV-68 infection of NIH 3T3 cells, indicating a potential role for cellular miRNAs during MHV-68 infection. Our data will aid in the full exploration of the functions of γHV miRNAs