Katja R. Richert-Pöggeler

Induction of infectious petunia vein clearing (pararetro) virus from endogenous provirus in petunia

Infection by an endogenous pararetrovirus using forms of both episomal and chromosomal origin has been demonstrated and characterized, together with evidence that petunia vein clearing virus (PVCV) is a constituent of the Petunia hybrida genome. Our findings allow comparative and direct analysis of horizontally and vertically transmitted virus forms and demonstrate their infectivity using biolistic transformation of a provirus‐free petunia species. Some integrants within the genome of P.hybrida are arranged in tandem, allowing direct release of virus by transcription. In addition to known inducers of endogenous pararetroviruses, such as genome hybridization, tissue culture and abiotic stresses, we observed activation of PVCV after wounding. Our data also support the hypothesis that the host plant uses DNA methylation to control the endogenous pararetrovirus.

Insight into the evolution of the Solanaceae from the parental genomes of Petunia hybrida

Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n = 14) containing 32,928 and 36,697 protein-coding genes, respectively. The genomes reveal that the Petunia lineage has experienced at least two rounds of hexaploidization: the older gamma event, which is shared with most Eudicots, and a more recent Solanaceae event that is shared with tomato and other solanaceous species. Transcription factors involved in the shift from bee to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral colour patterns and pollination systems. The high-quality genome sequences will enhance the value of Petunia as a model system for research on unique biological phenomena such as small RNAs, symbiosis, self-incompatibility and circadian rhythms.

Detection of petunia vein-clearing virus: model for the detection of DNA viruses in plants with homologous endogenous pararetrovirus sequences

A number of cases of plant virus sequence integration into host plant genome have been reported. In at least two cases, endogenous pararetrovirus sequences are correlated strongly with subsequent episomal virus infection and there is circumstantial evidence that this also occurs for Petunia vein-clearing virus (PVCV). The detection of viruses is a critical component of plant health and therefore, it is important to have diagnostic procedures that differentiate between the detection of encapsidated viral DNA and homologous sequences in the host genome. PCR-based detection methods targeted at PVCV DNA have been tested and particular attention was paid to design controls that would indicate the existence of host DNA in the reaction. The use of ion-exchange chromatography for the partial purification of plant viruses from other cellular components, including chromosomal DNA, is described. The methods tested for PVCV detection are used to illustrate general principles for the specific detection of virus infections in host plants that carry homologous virus sequences in their genomes.

Ortervirales: a new viral order unifying five families of reverse-transcribing viruses

Reverse-transcribing viruses, which synthesize a copy of genomic DNA from an RNA template, are widespread in animals, plants, algae and fungi (1, 2).….

First Report of physostegia chlorotic mottle virus on Tomato (Solanum lycopersicum) in Germany

In September 2015, a tomato sample collected in the German state of Hesse was sent to the Julius Kuhn-Institut for analysis. While the fruits showed marbling and discoloration, the leaf samples from this plant did not show any obvious symptoms. Transmission electron microscopy (TEM) revealed the presence of bullet-shaped virus particles indicating the presence of a rhabdovirus. However, immunosorbent electron microscopy using antiserum JKI-1073 for Eggplant mottled dwarf virus (EMDV) could not confirm EMDV infection. The virus was mechanically transmitted to Nicotiana benthamiana, N. clevelandii, and Chenopodium quinoa inducing yellowing and leaf deformation, while mechanical transmission to N. occidentalis (P1 and 37b) failed. Extraction of double stranded-RNA (dsRNA) followed by random-PCR (Froussard 1992), cloning of PCR products, and sequencing failed to reveal any virus sequences. Total RNA was extracted from infected N. benthamiana, followed by ribo-depletion, library preparation and submission for next-generation sequencing (NGS) using an Illumina MiSeq platform as described by Knierim et al. (2017). De novo assembly of the trimmed reads was done with Geneious v 10.1.3 (Biomatters LTD, NZ). Using MEGA BLAST, 13 contigs showed between 95.6 and 98.5% similarity with physostegia chlorotic mottle virus (PhCMoV) isolate PV-1182 (accession no. KX636164). The complete PhCMoV genome (13,321 nt length) was assembled by mapping reads to this reference genome and used to design PhCMoV-specific RT-PCR primers for detection (HZ-343 5′-CGGTGAGTGGGGCAACTAAT-3′/HZ-344 5′-AGCGATGGGGTCTAGTGTCT-3′). RT-PCR confirmed the presence of PhCMoV in the test plants resulting in amplicons of approximately 875 bp. In August 2016, similar symptoms on tomato fruits were observed by a different grower in Hesse. The presence of PhCMoV was confirmed by TEM and RT-PCR. Additionally, the PCR products were sequenced and showed 97% identity to KX636164. Surprisingly, reanalysis of a tomato sample from 2003 that was infected by a hitherto unknown rhabdovirus using NGS also confirmed infection with PhCMoV. This sample also originated from Hesse although the original grower is unknown. The complete genome of the 2003 PhCMoV sample was assembled following the same methods described above. Pairwise comparison between the genomes of 2015 and 2003 isolates resulted in 99.7% nucleotide identity and 96.9% when compared with KX636164. These findings indicate the presence of PhCMoV in tomato in Germany for a long time albeit isolated occurrences in different production areas. PhCMoV was recently identified from Physostegia virginiana plants showing leaf deformation and severe chlorotic and mottle symptoms in Austria (Menzel et al. 2016). However, it is not known if there is a link between PhCMoV isolates infecting P. virginiana and tomato as the routes of transmission and dissemination are currently unknown. The sequences from this report were deposited in GenBank (accession nos. KY706238 and KY859866 [full-length sequences], KY882263 and KY882264 [partial sequences]). To our knowledge, this is the first host record of PhCMoV in tomato and a new country record for Germany.

Charakterisierung eines Nucleorhabdovirus aus Physostegia

Die Daten der Vergleichsbetriebe stellen den Status quo im Pflanzenschutz in Deutschland dar. Der Behandlungsindex in Mohre und Frischkohl betrug im Durchschnitt der Jahre (2007-2014) bei 6,2 bzw. 9,2. Mohren wurden vorrangig mit Herbiziden und Frischkohl mit Insektiziden behandelt. Unter dem Aspekt des Pflanzenschutzes und zur Reduktion der Behandlungsintensitat mit Insektiziden kann Abbildung 1: Behandlungsindex der Herbizide (Herbizid-BI) in Abhangigkeit der Vorfrucht in den Vergleichsbetrieben fur Mohre, 2007-2014, in Deutschland, ungleiche Buchstaben symbolisieren signifikante Unterschiede (p<0,05), nBlattfrucht=58, nGemuse=28, nHalmfruchte=115 208 72. ALVA-Tagung, Seminarhotel Wesenufer, 2017 der Fruhkohlanbau empfohlen werden. Im Kohl- und Mohrenanbau konnen Halmvorfruchte Schadlingsbefall vorbeugen. Die Mittelaufwandmengen wurden vorrangig bei Herbizidanwendungen reduziert In beiden Kulturen wurden Abweichungen vom notwendigen Mas verzeichnet. Die Pflanzenschutzberatung sowie die regelmasige Durchfuhrung von Bonituren konnen Pflanzenschutzmittelanwendungen dem notwendigen Mas naher bringen und die Umsetzung des IPS in der Praxis verbessern. Fur die Ausschopfung nichtchemischer Masnahmen sind Praxisdemonstrationen und der intensive Austausch zwischen Wissenschaft und Praxis notig, genauso wie Forschungsforderung zur Entwicklung neuer und Optimierung bestehender integrierter Verfahren und neuer Wirkstoffe. Vorbildhaft wird der IPS von den Demonstrationsbetrieben u. a. in den Kulturen Mohre und Weiskohl, seit 2014 umgesetzt. Die Betriebe werden individuell und intensiv betraten, um Pflanzenschutzmittelanwendungen auf das notwendige Mas zu beschranken. Hierfur werden Bonituren unter Nutzung von Schadschwellen, Warndiensthinweisen und Monitoringverfahren durchgefuhrt.

Impact of Retroelements in Shaping the Petunia Genome

Retroelements, defined by their dependence on reverse transcription for replication, are found in the genomes of bacteria, fungi, animals and plants. This chapter summarizes current knowledge about the structure, function and evolution of representatives from two retroelement groups identified in Petunia. The presence of both a viral retroelement – an inducible endogenous plant pararetrovirus, EPRV- and non-viral retroelements in the form of LTR-retrotransposons makes Petunia an ideal model system to study possible retroelement interactions. Phylogenetic relationships have been determined and chromosomal co-localization of EPRV and Metaviridae, one group of LTR-retrotransposons, has been demonstrated. The impact of partly overlapping replication pathways on element interference is discussed. While studies in Petunia and related species have led to tremendous progress in our understanding of these elements we are just beginning to comprehend the consequences of their presence and activities in their hosts.