Abstract
Cucumber green mottle mosaic virus (CGMMV) is one of the most devastating viruses infecting members of the family Cucurbitaceae. The assembly initiation site of CGMMV is located in the coding region of the coat protein, which is not only involved in virion assembly but is also a key factor determining the long-distance movement of the virus. To understand the effect of assembly initiation site and the adjacent region on CGMMV infectivity, we created a GTT deletion mutation in the GAGGTTG assembly initiation site of the infectious clone of CGMMV, which we termed V97 (deletion mutation at residue 97 of coat protein), followed by the construction of the V94A and T104A mutants. We observed that these three mutations caused mosaic after Agrobacterium-mediated transformation in Nicotiana benthamiana, albeit with a significant delay compared to the wild type clone. The mutants also had a common spontaneous E96K mutation in the coat protein. These results indicated that the initial assembly site and the sequence of the adjacent region affected the infectivity of the virus and that E96 might play an essential role in this process. We constructed two single point mutants—E96A and E96K—and three double mutants—V94A-E96K, V97-E96K and T104A-E96K—to further understand the role of E96 in CGMMV pathogenesis. After inoculation in N. benthamiana, E96A showed delayed systemic symptoms, but the E96K and three double mutants exhibited typical symptoms of mosaic at seven days post-infection. Then, sap from CGMMV-infected N. benthamiana leaves was mechanically inoculated on watermelon plants. We confirmed that E96 affected CGMMV infection using double antibody sandwich-enzyme-linked immunosorbent assay (DAS-ELISA), reverse transcription-polymerase chain reaction (RT-PCR), and sequencing, which further confirmed the successful infection of the related mutants, and that E96K can compensate the effect of the V94, V97, and T104 mutations on virus infectivity. In addition, Northern blotting showed that the accumulation of viral RNA corroborated the severity of the symptoms.
Highlights
Cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, is a devastating virus infecting plants of the family Cucurbitaceae
Northern blotting was performed with the CGMMV RNA to understand the relationship between the accumulation of viral RNA and symptoms of the host plants
Total RNA was extracted from the plant virus coat protein (CP) possess multiple functions such as replication, virion formation, cell-to-cell upper uninoculated leaves of N. benthamiana using the Trizol reagent at 7 dpi
Summary
Cucumber green mottle mosaic virus (CGMMV), a member of the genus Tobamovirus, is a devastating virus infecting plants of the family Cucurbitaceae. It was first reported as cucumber virus 3 by Ainsworth in 1935 [1]. The assembly initiation site of CGMMV is located in the coding region of CP gene [22]. We assumed that the assembly initiation site and adjacent region might be correlated with virus infectivity. We mutated the assembly initiation site on the infectious cDNA clone of CGMMV to analyze this association. Results showed that the assembly initiation site and its adjacent region and the 96th amino acid of CP affected CGMMV infectivity
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