Simultaneous mutations in multi-viral proteins are required for soybean mosaic virus to gain virulence on soybean genotypes carrying different R genes

Abstract

Background: Genetic resistance is the most effective and sustainable approach to the control of plant pathogens that are a major constraint to agriculture worldwide. In soybean, three dominant R genes, i.e., Rsv1, Rsv3 and Rsv4, have been identified and deployed against Soybean mosaic virus (SMV) with strain-specificities. Molecular identification of virulent determinants of SMV on these resistance genes will provide essential information for the proper utilization of these resistance genes to protect soybean against SMV, and advance knowledge of virus-host interactions in general.

Methodology/principal findings: To study the gain and loss of SMV virulence on all the three resistance loci, SMV strains G7 and two G2 isolates L and LRB were used as parental viruses. SMV chimeras and mutants were created by partial genome swapping and point mutagenesis and then assessed for virulence on soybean cultivars PI96983 (Rsv1), L-29 (Rsv3), V94-5152 (Rsv4) and Williams 82 (rsv). It was found that P3 played an essential role in virulence determination on all three resistance loci and CI was required for virulence on Rsv1- and Rsv3-genotype soybeans. In addition, essential mutations in HC-Pro were also required for the gain of virulence on Rsv1-genotype soybean. To our best knowledge, this is the first report that CI and P3 are involved in virulence on Rsv1- and Rsv3-mediated resistance, respectively.

Conclusions/significance: Multiple viral proteins, i.e., HC-Pro, P3 and CI, are involved in virulence on the three resistance loci and simultaneous mutations at essential positions of different viral proteins are required for an avirulent SMV strain to gain virulence on all three resistance loci. The likelihood of such mutations occurring naturally and concurrently on multiple viral proteins is low. Thus, incorporation of all three resistance genes in a soybean cultivar through gene pyramiding may provide durable resistance to SMV.

Figure 1. Schematic representation of three full-length infectious clones derived from SMV strains G7 and two G2 isolates, L and LRB.

(A). Genomic organization of wild parental viral genomes with their respective viral proteins. Nucleotide position numbers for predicted mature proteins are indicated: italic for G7 and normal for L or LRB. (B). Restriction maps of G7 (top) and L or LRB (bottom). Arrows indicate the division of three cDNA fragments N, M and C and their nucleotide lengths are provided in parentheses. The length of the nucleotides between enzyme sites is given between those sites.

Figure 2. Pathogenicity assays of parental SMV infectious clones L, LRB and G7, chimeric clones and mutants with rsv-, Rsv1-, Rsv3- and Rsv4 -genotype soybeans.

The infectivity of the clones is shown to the right. rsv, Williams 82 (carrying no resistance gene); Rsv1, PI96983; Rsv3, L29; Rsv4, V94-5152; +, positive in ELISA and RT-PCR assays; –, negative in ELISA and RT-PCR assays.

Figure 3. Infectivity and symptoms of soybean inoculated with chimeric SMV clones G7(L 2339-4624) and G7(L 3625-4624).

(A). Inoculated with G7(L 2339-4624). Photos were taken 28 days post inoculation. (B). Inoculated with G7(L 3625-4624). Trifoliate leaves are shown underneath. Phtos were taken 21 days post inoculation. Symptoms are evident on rsv- and Rsv3-genotype soybeans. rsv, Williams 82 (carrying no resistance gene); Rsv1, PI96983; Rsv3, L29; Rsv4, V94-5152; +, positive (ELISA and RT-PCR); –, negative (ELISA and RT-PCR).

Figure 4. Amino acid sequence alignment of the SMV HC-Pro protein.

Arrow indicates restriction sites BglII and KpnI which were used for plasmid construction. Numbers are the amino acid positions of the deduced polyprotein encoded by the long open reading frame. As shown, the last 30 amino acid sequence (after KpnI) is identical between G7 and G2 strains. The position numbers of two point mutations in this study are indicated.

Figure 5. Amino acid sequence alignment of the SMV P3 protein.

Restriction site SpeI used for clone construction is shown. * indicates two essential amino acids K and R responsible for breaking down Rsv4-mediated resistance.

Figure 6. Infectivity and symptoms of soybean inoculated with chimeric SMV clones L(G7 2342-3237) and L(G7 2342-3237)(G1054R).

(A). inoculated with L(G7 2342-3237). (B). Inoculated with L(G7 2342-3237)(G1054R). Trifoliate leaves are shown underneath. Photos were taken 14 days post inoculation. rsv, Williams 82 (carrying no resistance gene); Rsv1, PI96983; Rsv3, L29; Rsv4, V94-5152; +, positive (ELISA and RT-PCR); –, negative (ELISA and RT-PCR).

Figure 7. Infectivity and symptoms of soybean inoculated with chimeric SMV clones L(G7 1608-3237) and L(G7 1608-3237)(G1054R).

(A). Inoculated with L(G7 1608-3237). (B). Inoculated with L(G7 1608-3237)(G1054R). Trifoliate leaves are shown underneath. Photos were taken 14 days post inoculation. rsv, Williams 82 (carrying no resistance gene); Rsv1, PI96983; Rsv3, L29; Rsv4, V94-5152; +, positive (ELISA and RT-PCR); –, negative (ELISA and RT-PCR).

Figure 8. Infectivity and symptoms of soybean inoculated with chimeric SMV clones L(G7 1-3237) G1054R and G7(L1-1605)L(3234-4624) G1054R.

(A). Inoculated with L(G7 1-3237) G1054R. (B). Inoculated with G7(L1-1605)L(3234-4624) G1054R. Trifoliate leaves are shown underneath. Photos were taken 3 weeks post inoculation. rsv, Williams 82 (carrying no resistance gene); Rsv1, PI96983; Rsv3, L29; Rsv4, V94-5152; +, positive (ELISA and RT-PCR); –, negative (ELISA and RT-PCR).

Figure 9. Symptoms of Rsv1-genotype soybean inoculated with SMV clones G7, L(G7 1608-3237), L(G7 1608-3237)(G1054R) and L(G7 2342-3237)(R682M, C720Y, G1054R).

Dead leaf tissues resulting from lethal systemic hypersensitive response (LSHR) were evident on Rsv1-genotype soybean inoculated with all four SMVs. Photos were taken 42 days post inoculation.

Figure 10. Infectivity and symptoms of soybean inoculated with chimeric SMV clones L(G7 2342-3237)(C720Y) and L(G7 2342-3237)(R682M, C720Y).

(A). Inoculated with L(G7 2342-3237)(C720Y). (B). Inoculated with L(G7 2342-3237)(R682M, C720Y). Trifoliate leaves are shown underneath. Photos were taken 14 days post inoculation. rsv, Williams 82 (carrying no resistance gene); Rsv1, PI96983; Rsv3, L29; Rsv4, V94-5152; +, positive (ELISA and RT-PCR); –, negative (ELISA and RT-PCR).

Figure 11. Infectivity and symptoms of soybean inoculated with chimeric SMV clones L(R682M C720Y) and LRB (R682M C720Y).

(A). Inoculated with L(R682M C720Y). (B). Inoculated with LRB (R682M C720Y). Trifoliate leaves are shown underneath. Photos were taken 3 weeks post inoculation. rsv, Williams 82 (carrying no resistance gene); Rsv1, PI96983; Rsv3, L29; Rsv4, V94-5152; +, positive (ELISA and RT-PCR); –, negative (ELISA and RT-PCR).