Barley stripe mosaic virus-encoded proteins triple-gene block 2 and γb localize to chloroplasts in virus-infected monocot and dicot plants, revealing hitherto-unknown roles in virus replication

Abstract

Replication of Barley stripe mosaic virus (BSMV), genus Hordeivirus, is thought to be associated with vesicles in proplastids and chloroplasts, but the molecular details of the process and identity of virus proteins involved in establishing the virus replication complexes are unknown. In addition, BSMV encodes a triple-gene block of movement proteins (TGBs) that putatively share functional roles with their counterparts in other hordei-, pomo-and pecluviruses, but detailed information on the intracellular locations of the individual TGBs is lacking. Here, the subcellular localizations of BSMV-encoded proteins TGB2 and cb fused to green or red fluorescent proteins were examined in epidermal cells of Nicotiana benthamiana and barley (Hordeum vulgare 'Black Hulless'). The fusion proteins were expressed from a BSMV vector or under the control of the cauliflower mosaic virus 35S promoter. The subcellular localizations were studied by confocal laser-scanning microscopy (CLSM). CLSM studies showed that both proteins were recruited to chloroplasts in the presence of viral RNA and that virus RNA, coat protein and cb protein were detected in plastid preparations from infected leaves. Electron microscope images of thin sections of virus-infected leaves revealed abnormal chloroplasts with cytoplasmic inclusions containing virus-like particles. In addition, cellular localizations of BSMV TGB2 suggest subtle differences in function between the hordei-like TGB2 proteins. The results indicate that TGB2 and cb proteins play a previously unknown functional role at the site of virus replication.

Mutation of a chloroplast-targeting signal in Alternanthera mosaic virus TGB3 impairs cell-to-cell movement and eliminates long-distance virus movement

“…Several reports describe virus interactions with chloroplasts, mainly relating to virus replication (Prod'homme et al, 2003;Torrance et al, 2006). Nevertheless, we show here for the first time that AltMV TGB3 is involved in transport between different cell types adjunct to replication.…”

Subcellular Localization of the Barley Stripe Mosaic Virus Triple Gene Block Proteins

“…The available evidence also indicates that hordeivirus-like and potexvirus-like TGB1 proteins share common biochemical features, including RNA binding abilities (3,13,23,35,44,56), RNA helicase activities (22), associated NTPase activities (3,13,23,33,35,44), and the capacity to form homologous interactions (29,30,45). However, the potexvirus-like TGB1 proteins localize at the CW when expressed autonomously and also facilitate increases in PD size exclusion limits, whereas the hordeivirus-like TGB1 proteins lack both these activities (39,53). Major differences are also evident in the organizations of the potexvirus-like and hordeivirus-like TGB3 proteins, which share no discernible relatedness, differ in the numbers of their transmembrane domains, and indeed appear to have a polyphyletic origin (39).…”

“…Viruses containing triple gene block (TGB) MPs have been the subjects of a number of investigations (4,6,39,53,54). Interestingly, viruses with a range of diverse genome structures encode MPs in a TGB, but these proteins fall into two major TGB classes that have substantial differences in protein structure and variations in their physical, functional, and cellular interactions (19,30,39,45,48).…”

Development of a one-step immunocapture real-time RT-PCR assay for the detection of barley stripe mosaic virus strains in barley seedlings

“…So far, mainly serological tests like ELISA (Faris-Mukhayyish and Makkouk 1983;Torrance 1998), immunogold technique (Hsu, 1984), filter paper sero-assay (FiPSA) (Haber and Knapen, 1989) and only few molecular techniques for BSMV detection (Estabrook at al., 1998;Torrance at al., 2006;Ay et al, 2008) have been published. All of these publications refer to aggressive isolates of BSMV.…”

“…The RNA was measured using a NanoDrop spectrophotometer (ThermoScientific) and adjusted to 50 ng/μl. Isolated RNA was subsequently used in RT-PCR with primers designed by Estabrook (1998) and Torrance (2006). A new primer set TGB2F (5'-GGATGAAGACCACAGTTGGTTC-3') and TGB2R (5'-CTAGCCAATATCGCATAGTAATG-3') was designed using the Oligo 6.0 software based on the alignment of the TGB2 gene of different BSMV sequences retrieved from the GenBank Database.…”