Characterization of a hypovirulent Chrysovirus from Fusarium graminearum: processing of the viral proteins, replication and infection
Plant Breeding and Plant Pathology
Final Report Abstract
The original strain infected with the chrysovirus FgV-ch9 is hypovirulent when infected on wheat and maize. The sequence of the five segments of the virus, each coding for one protein, was known. Three of the proteins were known to be part of the particle, two as coat proteins in a processed form and the RNA dependent RNA polymerase, while the other two were not found inside the particle. Starting from these facts time course studies revealed that the processing of the two envelope proteins was a coordinated process comprising several steps. Expression of all open reading frames (ORFs) in E. coli, S. cerevisiae and F. graminearum did not indicate, whether the processing mechanism were autoproteolytic, regulated by viral or host proteins. However, a C-terminal fusion with eGFP showed partly processing in E. coli and S. cerevisiae. The expression of the ORF of segment 5 revealed a higher molecular weight as proposed by the sequence. Resequencing of parts of this segment showed an internal duplication. This led to an increase of the molecular weight and to a duplication of the number of zinc fingers. Former results of the gene silencing suppression activity in Nicotiana benthamiana leaves were confirmed. In addition, the gene silencing suppressor activity was shown in F. graminearum. Resequencing of the 3´ termini of the segments 2 and 3 resulted again in duplications, which do not affect the ORFs. Computer analysis of the sequences of the closely related FgV2 virus and other chrysoviruses revealed similarly duplicated sequences to be present. A requirement for working with the virus FgV-ch9 is that the infection of strains and mutants by anastomosis formation was established. Virus infection of the wild-type strain FgPH1 resulted in the same restricted growth in complete medium and hypovirulence when infected on wheat. The infection of the wild-type strain of Fg8.1 failed, however, the infection with a deoxyhypusine hydroxylase (DOHH) overexpression, which has a reduced conidiation, was successful. When testing single conidia cultures originated from virus infected FgPH1 wildtype in only 10 % of the cultures a high virus titer could be detected. In contrast, the single conidia cultures of a FgPH1 mutant with a deletion of the Hex1 gene, a major component of the Woronin bodies, resulted in 100 % cultures with a high virus titer. The infection of a mutant, fd1oe, which overexpresses an mRNA binding protein, did not result in restricted growth or hypovirulence, while the knock-out of this gene resulted in restricted growth and loss of infection. A reduced growth of the mycelium and hypovirulence occurred again after the overexpression of the ORF of segment 3, the putative outer shell protein of the viral particle. In this mutant as well as in the virus infected wild-type strain the fd1 gene is highly down-regulated. Down-regulation by application of in vitro synthesized dsRNA of fd1 resulted in a reduction of mycelial growth. These results led to the idea of host-induced gene silencing of fd1.
Publications
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(2016) EP15170150.5 "Method of conferring resistance against a Fusarium plant disease"
Bormann J, Heinze C, Schäfer W
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(2017) Duplications in the 3´ -termini of three segments of Fusarium graminearum virus China 9. Archives of Virology 3: 897-900
Blum C, Götsch S, Heinze C
