A cucumber mosaic virus mutant lacking the 2b counter-defence protein gene provides protection against wild-type strains

Abstract

Several plant virus mutants, in which genes encoding silencing suppressor proteins have been deleted, are known to induce systemic or localized RNA silencing against themselves and other RNA molecules containing homologous sequences. Thus, it is thought that many cases of cross-protection, in which infection with a mild or asymptomatic virus mutant protects plants against challenge infection with closely related virulent viruses, can be explained by RNA silencing. We found that a cucumber mosaic virus (CMV) mutant of the subgroup IA strain Fny (Fny-CMVD2b), which cannot express the 2b silencing suppressor protein, cross-protects tobacco (Nicotiana tabacum) and Nicotiana benthamiana plants against disease induction by wild-type Fny-CMV. However, protection is most effective only if inoculation with Fny-CMVD2b and challenge inoculation with wild-type CMV occurs on the same leaf. Unexpectedly, FnyCMVD2b also protected plants against infection with TC-CMV, a subgroup II strain that is not closely related to Fny-CMV. Additionally, in situ hybridization revealed that Fny-CMVD2b and Fny-CMV can co-exist in the same tissues but these tissues contain zones of Fny-CMVD2b-infected host cells from which Fny-CMV appears to be excluded. Taken together, it appears unlikely that cross-protection by Fny-CMVD2b occurs by induction of systemic RNA silencing against itself and homologous RNA sequences in wild-type CMV. It is more likely that protection occurs through either induction of very highly localized RNA silencing, or by competition between strains for host cells or resources. INTRODUCTIONCucumber mosaic virus (CMV) is the type species of the genus Cucumovirus of the family Bromoviridae (Van Regenmortel et al., 2000). Based on serological relationships and sequence criteria, the species is divided into three distinct subgroups: IA, IB and II (Roossinck et al., 1999). CMV has the largest host range of any known plant virus and is transmitted in a non-persistent manner by aphids belonging to over 80 species. Taken together, these factors probably contribute to the worldwide distribution of the virus and its economic importance (Palukaitis et al., 1992;Palukaitis & García-Arenal, 2003). The CMV genome consists of three positive-sense RNA molecules. These are RNAs 1, 2 and 3, which also function as mRNAs for the synthesis of the 1a and 2a replicase proteins, and the 3a movement protein, respectively. During replication, subgenomic mRNAs encoding additional proteins are synthesized. The subgenomic RNA 4, derived from RNA 3, is the mRNA for CMV coat protein (CP) and RNA 4A, which is derived from RNA 2, is the mRNA for the multifunctional 2b protein (Ding et al., 1994). Direct control of CMV or prevention of its transmission by aphids by using insecticides is difficult to achieve. One promising approach for controlling CMV is the use of pathogen-derived transgenes in genetically modified plants (Palukaitis & Zaitlin, 1997;Gaba et al., 2004). Pathogenderived resistance to CMV and other viruses works either through the triggering of...

Effects of dicer-like endoribonucleases 2 and 4 on infection of Arabidopsis thaliana by cucumber mosaic virus and a mutant virus lacking the 2b counter-defence protein gene

“…Although CMVD2b protected to some extent against subsequent infection with Fny-CMV and another CMV strain, there was no indication that this mutant induced a strong, systemic silencing signal (Ziebell et al, 2007). In situ hybridization showed that, although both strains occurred in doubly infected leaves, they did not co-infect the same cells (Ziebell et al, 2007). Such spatial separation (exclusion) has been described for mixed infections between several virus strains (Dietrich et al, 2007;Hull & Plaskitt, 1970;Takahashi et al, 2007;Takeshita et al, 2004).…”

“…1, row 1, column 2), whereas Fny-CMV caused severe stunting, crinkling and deformation of the youngest plant tissue, resulting in a compact appearance (Fig. 1, row 1, column 4) (Lewsey et al, 2009;Ziebell et al, 2007). FnyCMVD2b was able to provide some cross-protection Fig.…”

“…We explored this concept by using a mutant of the Fny strain of cucumber mosaic virus (Fny-CMV) lacking the 2b silencing-suppressor protein gene (Fny-CMVD2b), reasoning that this deletion mutant might induce silencing against RNA sequences shared with the wild-type virus (Ziebell et al, 2007). Although CMVD2b protected to some extent against subsequent infection with Fny-CMV and another CMV strain, there was no indication that this mutant induced a strong, systemic silencing signal (Ziebell et al, 2007). In situ hybridization showed that, although both strains occurred in doubly infected leaves, they did not co-infect the same cells (Ziebell et al, 2007).…”

“…1, row 4, column 3). Therefore, plants were analysed by using RT-PCR to discriminate between wildtype and mutant CMV (Ziebell et al, 2007). Between six and 11 plants (out of a total of 12-40 plants per mutant or wild-type line) that had been inoculated sequentially with Fny-CMVD2b followed by Fny-CMV (Fig.…”

“…In some cases, a previously described RT-PCR assay (Ziebell et al, 2007) was used to detect and distinguish between the RNAs 2 of Fny-CMVD2b and Fny-CMV, using systemically infected leaf tissue for RNA extraction. When it was not necessary to distinguish between the two virus strains, an ELISA was used.…”