The Coat Protein Gene is Essential for the Systemic Infection of Cucumber Mosaic Virus in Cucumis figarei at a High Temerature.

Abstract

Systemic infection of cucumber mosaic virus (CMV) in Cucumis figarei at high temperature was investigated using reassortants and chimeric RNAs. Three CMV strains of pepo-, SO-, MY17-and Y-CMV were used: pepo-, SO-and MY17-CMV systemically infected C. figarei at 36•Ž, whereas Y-CMV did not as previously described. Inoculation with in vitro transcripts from biologically active cDNA clones of Y-CMV and pepo-CMV RNAs 1, 2 and 3 indicated that only the inocula containing pepo-CMV RNA3 induced systemic infection at 36•Ž, suggesting that the resistance against systemic infection by Y-CMV and its breakage by pepo-CMV were mapped to CMV RNA3. Inoculation with transcripts from the cDNA clones of Y-CMV RNAs 1, 2 and RNA3 of SO-CMV or MY17-CMV provoked systemic infection at high temperature, indicating that the systemic infection by SO-CMV or MY17-CMV was also mapped to RNA3. Analysis with chimeras constructed between cDNA clones of Y-CMV and pepo-CMV RNA 3 showed that the resistance and resistance breakage were mapped to the coat protein gene on CMV RNA3. Comparison of the nucleotide sequence of pepo-, SO-and MY17-CMV RNA3 with that of Y-CMV RNA3 revealed that the coat protein of Y-CMV differs from those of pepo-, SO-and MY17-CMV in four amino acids at positions 17 (Leu to Pro), 25 (Ser to Pro), 28 (Ser to Ala) and 129 (Ser to Pro). These results suggested that all or some of the four amino acids play an important role in determining long-distance movement of CMV in C. figarei at a high temperature.

Shoot meristem tissue of tobacco inoculated with Cucumber mosaic virus is infected with the virus and subsequently recovers from infection by RNA silencing

“…Small RNAs were separated on 15% polyacrylamide gels containing 7 M urea and transferred by electroblotting onto a Hybond-N ϩ membrane (Amersham Biosciences, Piscataway, NJ, USA). Hybridization of antisense siRNA corresponding to CMV RNAs was performed using DIG-labeled full-length CMV RNAs transcripts complementary to the CMV minusstranded RNAs obtained from the pepo-CMV clones (Saitoh et al 1999). An oligo DNA (5Ј-GGATCCTTT CAGAAAGCACCTTCC-3Ј) of about 25 nt, labeled with DIG using a DIG Oligonucleotide Tailing Kit (Roche Diagnostics), was used as a low-molecular-weight DNA marker.…”

“…After washing with DW and 2X SSC (1X SSC is 0.15 M NaCl plus 0.015 M sodium citrate), sections were covered with a prehybridization solution (50% formamide, 300 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM ethylenediaminetetraacetic acid, 1X Denhardt's solution, 0.25% sodium lauryl sulfate, 10% dextran sulfate) and incubated for 1 h at 42°C. After the prehybridization solution was removed, the sections were incubated in a hybridization solution containing a digoxygenin (Roche Diagnostics, Basel, Switzerland) (DIG)-labeled RNA probe complementary to the conserved 3Ј sequence of the four CMV plus-stranded RNAs (Saitoh et al 1999) for 12 h at 42°C. The sections were washed for 15 min at 50°C twice each with 2X SSC, 0.2X SSC, and 0.1X SSC, and incubated in a blocking solution for 60 min, followed by incubation in a 1 : 1000 dilution of alkaline phosphatase-conjugated anti-DIG antibody (Roche Diagnostics) for 30 min.…”

Studies on viral movement of Cucumber mosaic virus in infected plants

“…In an analysis with chimeric RNA 3 between pepo and Y, the factors were found to be in the coat protein (CP) gene. Comparison of the nucleotide sequences of the strains revealed that four amino acids, at positions 17, 25, 28 and 129, played an important role in the systemic infectivity at high temperature (Saitoh et al 1999).…”

The 2b protein of cucumber mosaic virus is essential for viral infection of the shoot apical meristem and for efficient invasion of leaf primordia in infected tobacco plants

“…Procedures for the preparation of shoot meristem and LP sections from inoculated tobacco plants, immunohistochemistry using anti-CMV IgG and in situ hybridization using a digoxigenin (DIG; Roche Diagnostics)-labelled RNA probe complementary to the conserved 39 untranslated region (39-UTR) sequence of the four CMV positive-sense RNAs (Saitoh et al, 1999) were conducted using methods described previously (Mochizuki & Ohki, 2004). The stained sections were examined with a BX-50 light microscope (Olympus).…”

“…The low-molecular-mass RNAs were transferred onto a Hybond-NX membrane (GE Healthcare Biosciences) by electroblotting. Hybridization was performed using DIG-labelled, full-length CMV RNAs complementary to the CMV negative-sense RNAs transcribed from Pepo CMV clones (Saitoh et al, 1999). The procedures for hybridization and detection of signals were performed as described previously (Mochizuki & Ohki, 2004).…”