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, 79 (2), 1215-22

Loss and Gain of Elicitor Function of Soybean Mosaic Virus G7 Provoking Rsv1-mediated Lethal Systemic Hypersensitive Response Maps to P3

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Loss and Gain of Elicitor Function of Soybean Mosaic Virus G7 Provoking Rsv1-mediated Lethal Systemic Hypersensitive Response Maps to P3

M R Hajimorad et al. J Virol.

Abstract

Rsv1, a single dominant resistance gene in soybean PI 96983 (Rsv1), confers extreme resistance against all known American strains of Soybean mosaic virus (SMV), except G7 and G7d. SMV-G7 provokes a lethal systemic hypersensitive response (LSHR), whereas SMV-G7d, an experimentally evolved variant of SMV-G7, induces systemic mosaic. To identify the elicitor of Rsv1-mediated LSHR, chimeras were constructed by exchanging fragments between the molecularly cloned SMV-G7 (pSMV-G7) and SMV-G7d (pSMV-G7d), and their elicitor functions were assessed on PI 96983 (Rsv1). pSMV-G7-derived chimeras containing only P3 of SMV-G7d lost the elicitor function, while the reciprocal chimera of pSMV-G7d gained the function. The P3 regions of the two viruses differ by six nucleotides, of which two are translationally silent. The four amino acid differences are located at positions 823, 915, 953, and 1112 of the precursor polypeptide. Analyses of the site-directed point mutants of both the viruses revealed that nucleotide substitutions leading to translationally silent mutations as well as reciprocal amino acid substitution at position 915 did not influence the loss or gain of the elicitor function. pSMV-G7-derived mutants with amino acid substitutions at any of the other three positions lost the ability to provoke LSHR but induced SHR instead. Two concomitant amino acid substitutions at positions 823 (V to M) and 953 (K to E) abolished pSMV-G7 elicitor function, provoking Rsv1-mediated SHR. Conversely, pSMV-G7d gained the elicitor function of Rsv1-mediated LSHR by a single amino acid substitution at position 823 (M to V), and mutants with amino acid substitutions at position 953 or 1112 induced SHR instead of mosaic. Taken together, the data suggest that strain-specific P3 of SMV is the elicitor of Rsv1-mediated LSHR.

Figures

FIG. 1.
FIG. 1.
Chimeric viruses and their phenotypic responses on soybean line PI 96983 (Rsv1). (A) Proposed genomic map of SMV (35). (B) Schematic representation of chimeric viruses constructed by exchanging fragments between pSMV-G7 (G7) and pSMV-G7d (G7d) utilizing the single restriction sites KpnI (Kp), SpeI (Sp), SalI (Sa), StuI (St), and ApaI (Ap) common between the two viruses. For construction of G7/G7d(1-2337) and G7d/G7(1-2337), the unique restriction site NotI located 669 nucleotides upstream of SMV sequences within vector sequences (data not shown) and the KpnI site were utilized. (C) Detection of progenies of the chimeric viruses in infected PI 96983 (Rsv1) soybean plants by slot blot hybridization. Following inoculation, plants were maintained in a growth chamber (20°C) until a leaflet from trifoliolates 3 and 4 of infected plants was collected 4 weeks postinoculation. Samples from corresponding trifoliolate leaflets of four independent replicate plants were combined; total RNA was isolated and denatured, and 10 μg was slot blotted onto a membrane and hybridized with 32P-labeled cDNA probes. (D) Phenotypic responses of soybean line PI 96983 (Rsv1) to mechanical inoculation with infectious sap containing progenies of parental or chimeric viruses. Following inoculation, the plants were maintained in a growth chamber (20°C) until virus-induced symptoms, mosaic or LSHR, were recorded about 6 weeks postinoculation.
FIG. 2.
FIG. 2.
Phenotypic differences in responses of soybean line PI 96983 (Rsv1) to inoculation with infectious sap containing progenies of pSMV-G7 (G7), pSMV-G7d (G7d), or their derivative chimeras. Following inoculation, the plants were maintained in a growth chamber (20°C) until photographed about 6 weeks postinoculation.
FIG. 3.
FIG. 3.
Genetic differences between P3 of pSMV-G7 and that of pSMV-G7d. (A) Proposed genomic map of SMV (35) indicating the size and the position of P3. (B) Nucleotide and amino acid differences between P3 of pSMV-G7 and that of pSMV-G7d. The positions of nucleotides and amino acids on the SMV genomes are based on sequences of SMV strains G7 and G7d (GenBank accession no. AY216010 and AY216987, respectively). Numbers in parentheses show positions of nucleotides within the codon.
FIG. 4.
FIG. 4.
Phenotypic differences in responses of soybean line PI 96983 (Rsv1) to inoculation with sap containing progenies of pSMV-G7 (G7), pSMV-G7d (G7d), or their derivative point mutants generated by site-directed mutagenesis of their respective P3. Following inoculation, the plants were maintained in a growth chamber (20°C) until photographed about 6 weeks postinoculation.
FIG. 5.
FIG. 5.
Slot blot hybridization analysis of accumulation of SMV RNA, soybean PR-1 protein gene transcript up regulation (PR1), and soybean 18S rRNA in soybean trifoliate leaves. Primary leaves of PI 96983 (Rsv1) were mechanically inoculated with buffer (mock) or infectious sap containing progenies of pSMV-G7 (G7), pSMV-G7d (G7d), or their derivative point mutants generated by site-directed mutagenesis. Following inoculation, plants were maintained in a growth chamber (20°C) until a leaflet from trifoliolates 3 and 4 of infected plants was collected about 6 weeks postinoculation. Samples from corresponding trifoliolate leaflets of four independent replicate plants were combined, total RNA was isolated and denatured, and 10 μg was slot blotted onto a membrane and hybridized with 32P-labeled cDNA probes.

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