TABLE 1.
Systemic infection of rsv1 (susceptible) and Rsv1 (functionally immune) soybean genotypes following biolistic or mechanical inoculation with mutant virusesa
| Mutantb | Systemic infection rate by genotype, cultivar, and infection method (no. of plants infected/no. of plants inoculated)a |
|||||||||
|---|---|---|---|---|---|---|---|---|---|---|
|
rsv1 |
Rsv1 |
|||||||||
| Williams 82 |
Lee 68 |
L800 |
PI 96983 |
L78-379 |
||||||
| Biolistic | Mechanical | Biolistic | Mechanical | Biolistic | Mechanical | Biolistic | Mechanical | Biolistic | Mechanical | |
| SMV-NG319S | 3/3 | 4/4 | 6/6 | 1*/4 | 0/4 | 0/6 | 0/6 | |||
| SMV-NG319S+K321E | 3/3 | 4/4 | 6/6 | 1*/4 | 0/9 | 0/13 | 0/7 | |||
| SMV-NK321E | 3/3 | 8/8 | 6/6 | 0/4 | 1*/8 | 0/6 | 0/4 | |||
| SMV-NT341I | 3*/3 | 4/4 | 6/6 | 0/3 | 0/6 | 0/9 | 0/8 | |||
| SMV-NK321E+T341I | 3*/3 | 6/7 | 6/6 | 0/4 | 0/4 | 0/12 | 0/6 | |||
| SMV-NR682M | 3*/3 | 3/3 | 6/6 | 0/4 | 0/4 | 0/8 | 0/7 | |||
| SMV-NR787I | 3/3 | 4/4 | 6/6 | 0/3 | 1*/6 | 0/6 | 0/12 | |||
| SMV-NR945G | 3/3 | 2/3 | 5/5 | 2*/3 | 4/5 | 0/7 | 0/9 | |||
| SMV-NA947T | 3/4 | 2/2 | 5/5 | 2*/3 | 2/2 | 0/4 | 0/4 | |||
| SMV-NA947V | 3/3 | 7/7 | 6/6 | 3*/3 | 8/9 | 0/5 | 0/6 | |||
| SMV-NP948L | 3/3 | 7/7 | 7/7 | 1*/4 | 4/5 | 0/6 | 0/6 | |||
| SMV-NK952E | 3/3 | 3/4 | 6/6 | 0/3 | 1*/7 | 0/6 | 0/6 | |||
| SMV-NV1045A | 3/3 | 3/3 | 6/6 | 1*/3 | 4/5 | 0/8 | 0/8 | |||
| SMV-NM1157I | 4*/4 | 6/6 | 6/6 | 0/3 | 0/5 | 0/6 | 0/6 | |||
| SMV-NR787I+A947T | 3/3 | 5/6 | 5/5 | 2*/3 | 6/6 | 0/12 | 0/22 | |||
| SMV-NK321E+R945G | 3/3 | 3/3 | 6/6 | 2*/4 | 3/5 | 0/16 | 0/7 | |||
| SMV-NK321E+A947V | 3/3 | 6/6 | 6/6 | 3*/4 | 4/4 | 0/6 | 0/6 | |||
| SMV-NK321E+T341I+A947V | 3/3 | 6/6 | 6/6 | 2/4 | 5/6 | 4/4 | 5/6 | 3*/4 | 2/7 | |
| SMV-NK321E+T341I+A947VDsR | 3/3 | 3/3 | 4/4 | 4/4 | 3/4 | |||||
| SMV-NK321E+T341I+A947V+V1045A | 3/3 | 7/7 | 6/6 | 4/4 | 4/4 | 4/4 | 6/6 | 4*/4 | 14/14 | |
| SMV-NG319S+K321E+A947V | 3/3 | 3/3 | 7/7 | 4/4 | 2/6 | 4*/4 | 5/10 | 0/4 | 0/5 | |
| SMV-NG319S+K321E+A947V+K952E | 2/2 | 3/3 | 6/6 | 5/5 | 6/6 | 4/4 | 6/6 | 1*/4 | 7/7 | |
| SMV-NG319S+K321E+R682M+A947V+K952E+M1157I | 3/3 | 3/3 | 5/5 | 3/4 | 7/7 | 3/4 | 12/12 | 4*/4 | 4/5 | |
| SMV-NG319S+K321E+R682M+A947V+K952E+M1157IDsR | 3/3 | 3/3 | 4/4 | 4/4 | 4/4 | |||||
| SMV-NR682M+R787I+A947T | 3/3 | 2/2 | 11/11 | 3/4 | 2/2 | 4/4 | 5/7 | 4*/4 | 14/14 | |
| SMV-G7I788Rc | 3/3 | 6/8 | 13/15 | 3/4 | 2/5 | 3*/4 | 0/7 | |||
| SMV-G7dI788Rc | 3/3 | 8/8 | 6/6 | 4/4 | 5/5 | 6/6 | 4*/4 | |||
For biolistic inoculation, plasmids containing full-length infectious cDNA clones were delivered biolistically by a helium gene gun system into fully expanded primary leaves of 2-week-old soybean seedlings. For mechanical inoculations, infectious extract from biolistically inoculated Williams 82 (rsv1) was used as an inoculum and rub inoculated onto fully expanded primary leaves of 2-week old soybean seedlings. The inoculated plants were maintained in a growth chamber (22°C) until evaluated for infection based on symptom expression. Absence of virus in asymptomatic plants was confirmed by ELISA. The asterisk indicates that for each virus variant, total RNA was extracted from one infected plant of the indicated genotype and subjected to RT-PCR. The stability of the introduced mutation(s) and lack of any newly emerged substitution(s) in progeny viruses was confirmed by sequencing the entire HC-Pro and P3 cistrons. Infection of a single L800 (Rsv1) plant following direct biolistic inoculation with SMV-NG319S was associated with sequence polymorphism at nucleotide position 1114 where both C and T were present. Thus, codons CCT and CTT encoding proline and leucine, respectively, were both present at amino acid position 328. Infection of a single L800 (Rsv1) by SMV-NK321E upon mechanical inoculation was associated with a newly emerged silent mutation in HC-Pro cistron (A2174G). Infection of a single L800 (Rsv1) by SMV-NK952E upon mechanical inoculation was associated with a newly emerged silent mutation (A1271G) in HC-Pro and a sequence polymorphism in P3 at position 2709 where both TCA and ACA codons encoding serine and threonine, respectively, were present at polyprotein position 860. Infection of L78-379 (Rsv1) by SMV-G7dI788R was associated with a missense mutation in P3 (G2752T) resulting in a C874F substitution in the virus polyprotein. No newly emerged mutations in HC-Pro and P3 cistrons of the progeny derived from any of the other molecularly cloned mutant viruses described in this table and Fig. 2 were detected.
DsR, DsRedT4 expression was visualized with an Olympus fluorescence stereomicroscope.
Substitutions at position 788 in the genomes of SMV-G7 and SMV-G7d correspond to 787 in the genome of SMV-N. This is because of lack of a codon in P1 cistron of SMV-N, which is located upstream of the P3 cistron (14).