A new bunyaviral-type plant virus associated with the ringspot disease of European mountain ash (Sorbus aucuparia L.). Genome organisation and detection of viral RNA by in situ-Hybridisation (ISH)

Chlorotic ringspots and mottling symptoms on leaves from European mountain ash trees (Sorbus aucuparia L.) are found in many European countries. The agent of this disease is still unknown, but we found associated with symptomatic trees a negative-strand, bunyaviral-type plant RNA virus, which is not specified so far. We named this new virus tentatively European mountain ash ringspot associated virus (EMARAV). Virus presence in symptomatic leaves is indicated by detection and extraction of four dsRNAs of approximately 7 kb, 2.3 kb, 1.5 kb and 1.3 kb. In asymptomatic trees no dsRNA can be detected. Using random primed reverse transcription and DOP-PCR, dsRNA-specific cDNA fragments could be generated [1]. From these cDNA clones primers were derived and used for 5'-RACE analyses. With this strategy a cDNA of 7 kb was obtained. The according RNA contains one ORF with homologies to the RNA dependent RNA polymerase (RdRp) of members belonging to the virus family Bunyaviridae. It shows all conserved motifs characteristically for bunyaviral RdRps and the typical complementary terminal sequences at its ends. Interestingly, the homology of these termini to those of the Tospovirus group, the only plant pathogenic members of the Bunyaviridae, is lower as compared to the corresponding sequences of Orthobunyaviruses and Hantaviruses, pointing at a putative common ancestor. Primers deduced from the terminus sequences allowed to identify another three RNAs of 2.3 kb, 1.5 kb and 1.3 kb. The corresponding ORFs encode a putative glycoprotein precursor, a putative nucleocapsid protein and a protein whose function is still unclear.

In situ-hybridisation experiments using Dig-labelled RNA-probes for the viral RNA 1 and RNA 3 showed a cluster of virus in different tissues of leaves from symptomatic trees. Primarily, cells of the palisade parenchyma and the spongy parenchyma appear to be infected by the virus.

Actual studies concentrate on analyses of protein function and on quantification of viral RNA by real-time RT-PCR.

Fig. 1: European mountain ash leaves showing chlorotic mottling (left) and chlorotic ringspots (right).

Fig. 2: Genome organisation of EMARAVirus. The identified and sequenced viral RNAs correspond in size with the earlier detected dsRNA pattern of symptomatic (S) mountain ash during native gel electrophoresis. No dsRNA could be isolated from asymptomatic (AS) trees.

[1] Benthack W, Mielke N, Büttner C and Mühlbach H-P: „Double-stranded RNA pattern and partial sequence data indicate plant virus infection associated with the ringspot disease of European mountain ash (Sorbus aucuparia L.).“ Arch. Virol. 150. 37-52 (2005). [View]