Viral suppressor of RNA silencing in vascular plants also interferes with the development of the bryophyte Physcomitrella patens.

Plant viruses are important pathogens able to overcome plant defense mechanisms using their viral suppressors of RNA silencing (VSR). Small RNA pathways of bryophytes and vascular plants have significant similarities, but little is known about how viruses interact with mosses. This study elucidated the responses of Physcomitrella patens to two different VSRs. We transformed P. patens plants to express VSR P19 from tomato bushy stunt virus and VSR 2b from cucumber mosaic virus, respectively. RNA sequencing and quantitative PCR were used to detect the effects of VSRs on gene expression. Small RNA (sRNA) sequencing was used to estimate the influences of VSRs on the sRNA pool of P. patens. Expression of either VSR-encoding gene caused developmental disorders in P. patens. The transcripts of four different transcription factors (AP2/erf, EREB-11 and two MYBs) accumulated in the P19 lines. sRNA sequencing revealed that VSR P19 significantly changed the microRNA pool in P. patens. Our results suggest that VSR P19 is functional in P. patens and affects the abundance of specific microRNAs interfering with gene expression. The results open new opportunities for using Physcomitrella as an alternative system to study plant-virus interactions.

A vertically transmitted amalgavirus is present in certain accessions of the bryophyte Physcomitrium patens.

In the last few years, next-generation sequencing techniques have started to be used to identify new viruses infecting plants. This has allowed to rapidly increase our knowledge on viruses other than those causing symptoms in economically important crops. Here we used this approach to identify a virus infecting Physcomitrium patens that has the typical structure of the double-stranded RNA endogenous viruses of the Amalgaviridae family, which we named Physcomitrium patens amalgavirus 1, or PHPAV1. PHPAV1 is present only in certain accessions of P. patens, where its RNA can be detected throughout the cell cycle of the plant. Our analysis demonstrates that PHPAV1 can be vertically transmitted through both paternal and maternal germlines, in crosses between accessions that contain the virus with accessions that do not contain it. This work suggests that PHPAV1 can replicate in genomic backgrounds different from those that actually contain the virus and opens the door for future studies on virus-host coevolution.

Plant genomes enclose footprints of past infections by giant virus relatives.

Nucleocytoplasmic large DNA viruses (NCLDVs) are eukaryotic viruses with large genomes (100 kb-2.5 Mb), which include giant Mimivirus, Megavirus and Pandoravirus. NCLDVs are known to infect animals, protists and phytoplankton but were never described as pathogens of land plants. Here, we show that the bryophyte Physcomitrella patens and the lycophyte Selaginella moellendorffii have open reading frames (ORFs) with high phylogenetic affinities to NCLDV homologues. The P. patens genes are clustered in DNA stretches (up to 13 kb) containing up to 16 NCLDV-like ORFs. Molecular evolution analysis suggests that the NCLDV-like regions were acquired by horizontal gene transfer from distinct but closely related viruses that possibly define a new family of NCLDVs. Transcriptomics and DNA methylation data indicate that the NCLDV-like regions are transcriptionally inactive and are highly cytosine methylated through a mechanism not relying on small RNAs. Altogether, our data show that members of NCLDV have infected land plants.

Unique translation initiation at the second AUG codon determines mitochondrial localization of the phage-type RNA polymerases in the moss Physcomitrella patens.

The nuclear genome of the moss Physcomitrella patens contains two genes encoding phage-type RNA polymerases (PpRPOT1 and PpRPOT2). Each of the PpRPOT1 and PpRPOT2 transcripts possesses two in-frame AUG codons at the 5' terminus that could act as a translational initiation site. Observation of transient and stable Physcomitrella transformants expressing the 5' terminus of each PpRPOT cDNA fused with the green fluorescent protein gene suggested that both PpRPOT1 and PpRPOT2 are not translated from the first (upstream) AUG codon in the natural context but translated from the second (downstream) one, and that these enzymes are targeted only to mitochondria, although they are potentially targeted to plastids when translation is forced to start from the first AUG codon. The influence of the 5'-upstream sequence on the translation efficiency of the two AUG codons in PpRPOT1 and PpRPOT2 was quantitatively assessed using a beta-glucuronidase reporter. The results further supported that the second AUG codon is the sole translation initiation site in Physcomitrella cells. An Arabidopsis (Arabidopsis thaliana) RPOT homolog AtRpoT;2 that possesses two initiation AUG codons in its transcripts, as do the RPOTs of P. patens, has been regarded as a dually targeted protein. When the localization of AtRpoT;2 was tested using green fluorescent protein in a similar way, AtRpoT;2 was also observed only in mitochondria in many Arabidopsis tissues. These results suggest that, despite the presence of two in-frame AUGs at the 5' termini of RPOTs in Physcomitrella and Arabidopsis, the second AUG is specifically recognized as the initiation site in these organisms, resulting in expression of a protein that is targeted to mitochondria. This finding may change the current framework of thinking about the transcription machinery of plastids in land plants.

Quantitative promoter analysis in Physcomitrella patens: a set of plant vectors activating gene expression within three orders of magnitude.

BACKGROUND: In addition to studies of plant gene function and developmental analyses, plant biotechnological use is largely dependent upon transgenic technologies. The moss Physcomitrella patens has become an exciting model system for studying plant molecular processes due to an exceptionally high rate of nuclear gene targeting by homologous recombination compared with other plants. However, its use in transgenic approaches requires expression vectors that incorporate sufficiently strong promoters. To satisfy this requirement, a set of plant expression vectors was constructed and equipped with either heterologous or endogenous promoters. RESULTS: Promoter activity was quantified using the dual-luciferase reporter assay system. The eight different heterologous promoter constructs tested exhibited expression levels spanning three orders of magnitude. Of these, the complete rice actin1 gene promoter showed the highest activity in Physcomitrella, followed by a truncated version of this promoter and three different versions of the cauliflower mosaic virus 35S promoter. In contrast, the Agrobacterium tumefaciens nopaline synthase promoter induced transcription rather weakly. Constructs including promoters commonly used in mammalian expression systems also proved to be functional in Physcomitrella. In addition, the 5' -regions of two Physcomitrella glycosyltransferases (i.e. alpha1,3-fucosyltransferase and beta1,2-xylosyltransferase) were identified and functionally characterised in comparison to the heterologous promoters. Furthermore, motifs responsible for enhancement of translation efficiency - such as the TMV omega element and a modified sequence directly prior the start codon - were tested in this model. CONCLUSION: We developed a vector set that enables gene expression studies, both in lower and higher land plants, thus providing valuable tools applicable in both basic and applied molecular research.