BackgroundMicroRNAs (miRNAs) are regulatory RNA molecules that are specified by their mode of action, the structure of primary transcripts, and their typical size of 20–24 nucleotides. Frequently, not only single miRNAs but whole families of closely related miRNAs have been found in animals and plants. Some families are widely conserved among different plant taxa. Hence, it is evident that these conserved miRNAs are of ancient origin and indicate essential functions that have been preserved over long evolutionary time scales. In contrast, other miRNAs seem to be species-specific and consequently must possess very distinct functions. Thus, the analysis of an early-branching species provides a window into the early evolution of fundamental regulatory processes in plants.ResultsBased on a combined experimental-computational approach, we report on the identification of 48 novel miRNAs and their putative targets in the moss Physcomitrella patens. From these, 18 miRNAs and two targets were verified in independent experiments. As a result of our study, the number of known miRNAs in Physcomitrella has been raised to 78. Functional assignments to mRNAs targeted by these miRNAs revealed a bias towards genes that are involved in regulation, cell wall biosynthesis and defense. Eight miRNAs were detected with different expression in protonema and gametophore tissue. The miRNAs 1–50 and 2–51 are located on a shared precursor that are separated by only one nucleotide and become processed in a tissue-specific way.ConclusionOur data provide evidence for a surprisingly diverse and complex miRNA population in Physcomitrella. Thus, the number and function of miRNAs must have significantly expanded during the evolution of early land plants. As we have described here within, the coupled maturation of two miRNAs from a shared precursor has not been previously identified in plants.

Eukaryotic organisms have dynamic genomes, with transposable elements (TEs) as a major contributing factor. Although the large autonomous TEs can significantly shape genomic structures during evolution, genomes often harbor more miniature nonautonomous TEs that can infest genomic niches where large TEs are rare. In spite of their cut-and-paste transposition mechanisms that do not inherently favor copy number increase, miniature inverted-repeat transposable elements (MITEs) are abundant in eukaryotic genomes and exist in high copy numbers. Based on the large number of MITE families revealed in previous studies, accurate annotation of MITEs, particularly in newly sequenced genomes, will identify more genomes highly rich in these elements. Novel families identified from these analyses, together with the currently known families, will further deepen our understanding of the origins, transposase sources, and dramatic amplification of these elements.

Trans-acting small interfering RNAs (ta-siRNAs) are plant-specific siRNAs released from TAS precursor transcripts after microRNA-dependent cleavage, conversion into double-stranded RNA, and Dicer-dependent phased processing. Like microRNAs (miRNAs), ta-siRNAs direct site-specific cleavage of target RNAs at sites of extensive complementarity. Here, we show that the DICER-LIKE 4 protein of Physcomitrella patens (PpDCL4) is essential for the biogenesis of 21 nucleotide (nt) ta-siRNAs. In ΔPpDCL4 mutants, off-sized 23 and 24-nt ta-siRNAs accumulated as the result of PpDCL3 activity. ΔPpDCL4 mutants display severe abnormalities throughout Physcomitrella development, including sterility, that were fully reversed in ΔPpDCL3/ΔPpDCL4 double-mutant plants. Therefore, PpDCL3 activity, not loss of PpDCL4 function per se, is the cause of the ΔPpDCL4 phenotypes. Additionally, we describe several new Physcomitrella trans-acting siRNA loci, three of which belong to a new family, TAS6. TAS6 loci are typified by sliced miR156 and miR529 target sites and are in close proximity to PpTAS3 loci.

Tomato big bud was detected for the first time in tomato plants (Lycopersicon esculentum Mill.) in the eastern region (Al‐Mafraq) of Jordan. Infected plants showed proliferation of lateral shoots, hypertrophic calyxes and greening of flower petals. The presence of phytoplasmas in diseased tomato plants was demonstrated using polymerase chain reaction (PCR) assays. The amplified DNAs yielded products of 1.8 kb (primer pair P1/P7) and 1.2 kb (primer pair R16F2/R2) by direct and nested‐PCR, respectively. DNA from tomato isolates T1 and T2 could not be amplified in the nested‐PCR assays when the aster yellow‐specific primer pair R16(1)F1/R1 was used, suggesting that the phytoplasma in these isolates is not genetically related to the 16SrI (aster yellows) group. After restriction fragment length polymorphism (RFLP) analyses, using four endonuclease enzymes (HhaI, RsaI, AluI and Bsp143I) similar patterns were formed among the digested 1.2 kb PCR products of two tomato isolates suggesting that both isolates belonged to the same phytoplasma. Compared with the RFLP profile of the reference strains, no difference in the digestion pattern was found between the tomato isolates and that of the catharanthus phyllody agent from Sudan, indicating that the phytoplasma belongs to 16SrDNA VI (clover proliferation) group.

Aster yellows phytoplasma were detected, for the first time, in peach trees in Al-Jubiha and Homret Al-Sahen area. Leaves of infected trees showed yellow or reddish, irregular water-soaked blotches. Discoloured areas become dry and brittle and the dead tissues dropped out. Under severe infections, leaves fall down and fruits dropped prematurely. Phytoplasmas were detected from all symptomatic peach trees by polymerase chain reaction (PCR) using universal phytoplasmas primers P1/P7 followed by R16F2/R2. No amplification products were obtained from templates of asymptomatic peaches. PCR products (1.2 kb) used for restriction fragment length polymorphism analysis (RFLP) after digestion with endonuclease AluI, HpaII, KpnI and RsaI produced the same restriction profiles for all samples, and they were identical with those of American aster yellows (16SrI) phytoplasma strain. This paper is the first report on aster yellows phytoplasma affecting peach trees in Jordan.

Eukaryotic genomes contain numerous DNA transposons that move by a cut-and-paste mechanism. The majority of these elements are self-insufficient and dependent on their autonomous relatives to transpose. Miniature inverted repeat transposable elements (MITEs) are often the most numerous nonautonomous DNA elements in a higher eukaryotic genome. Little is known about the origin of these MITE families as few of them are accompanied by their direct ancestral elements in a genome. Analyses of MITEs in the yellow fever mosquito identified its youngest MITE family, designated as Gnome, that contains at least 116 identical copies. Genome-wide search for direct ancestral autonomous elements of Gnome revealed an elusive single copy Tc1/Mariner-like element, named as Ozma, that encodes a transposase with a DD37E triad motif. Strikingly, Ozma also gave rise to two additional MITE families, designated as Elf and Goblin. These three MITE families were derived at different times during evolution and bear internal sequences originated from different regions of Ozma. Upon close inspection of the sequence junctions, the internal deletions during the formation of these three MITE families always occurred between two microhomologous sites (6–8 bp). These results suggest that multiple MITE families may originate from a single ancestral autonomous element, and formation of MITEs can be mediated by sequence microhomology. Ozma and its related MITEs are exceptional candidates for the long sought-after endogenous active transposon tool in genetic control of mosquitoes.

Miniature inverted repeat transposable elements (MITEs) lack protein coding capacity and often share very limited sequence similarity with potential autonomous elements. Their capability of efficient transposition and dramatic amplification led to the proposition that MITEs are an untapped rich source of materials for transposable element (TE) based genetic tools. To test the concept of using MITE sequence in gene transfer, a rice Stowaway MITE previously shown to excise efficiently in yeast was engineered to carry cargo genes (neo and gfp) for delivery into the budding yeast genome. Efficient excision of the cargo gene cassettes was observed even though the excision frequency generally decreases with the increase of the cargo sizes. Excised elements insert into new genomic loci efficiently, with about 65% of the obtained insertion sites located in genes. Elements at the primary insertion sites can be remobilized, frequently resulting in copy number increase of the element. Surprisingly, the orientation of a cargo gene (neo) on a construct bearing dual reporter genes (gfp and neo) was found to have a dramatic effect on transposition frequency. These results demonstrated the concept that MITE sequences can be useful in engineering genetic tools to deliver cargo genes into eukaryotic genomes.

MicroRNAs (miRNAs) are ~21 nt long small RNAs transcribed from endogenous MIR genes which form precursor RNAs with a characteristic hairpin structure. miRNAs control the expression of cognate target genes by binding to reverse complementary sequences resulting in cleavage or translational inhibition of the target RNA. Artificial miRNAs (amiRNAs) can be generated by exchanging the miRNA/miRNA sequence of endogenous MIR precursor genes, while maintaining the general pattern of matches and mismatches in the foldback. Thus, for functional gene analysis amiRNAs can be designed to target any gene of interest. During the last decade the moss Physcomitrella patens emerged as a model plant for functional gene analysis based on its unique ability to integrate DNA into the nuclear genome by homologous recombination which allows for the generation of targeted gene knockout mutants. In addition to this, we developed a protocol to express amiRNAs in P. patens that has particular advantages over the generation of knockout mutants and might be used to speed up reverse genetics approaches in this model species.