Abstract
Previous results using a movement defective alfalfa mosaic virus (AMV) vector revealed that citrus leprosis virus C (CiLV-C) movement protein (MP) generates a more efficient local movement, but not more systemic transport, than citrus leprosis virus C2 (CiLV-C2) MP, MPs belonging to two important viruses for the citrus industry. Here, competition experiment assays in transgenic tobacco plants (P12) between transcripts of AMV constructs expressing the cilevirus MPs, followed by several biological passages, showed the prevalence of the AMV construct carrying the CiLV-C2 MP. The analysis of AMV RNA 3 progeny recovered from P12 plant at the second viral passage revealed the presence of a mix of progeny encompassing the CiLV-C2 MP wild type (MPWT) and two variants carrying serines instead phenylalanines at positions 72 (MPS72F) or 259 (MPS259F), respectively. We evaluated the effects of each modified residue in virus replication, and cell-to-cell and long-distance movements. Results indicated that phenylalanine at position 259 favors viral cell-to-cell transport with an improvement in viral fitness, but has no effect on viral replication, whereas mutation at position 72 (MPS72F) has a penalty in the viral fitness. Our findings indicate that the prevalence of a viral population may be correlated with its greater efficiency in cell-to-cell and systemic movements.
Highlights
Introduction published maps and institutional affilIn the course of evolution, plant viruses have developed a special class of proteins specialized in viral transport, called movement proteins (MPs) [1]
For analysis of alfalfa mosaic virus (AMV) RNA accumulation, protoplasts were extracted from P12 leaves and 2.5 × 105 protoplasts were inoculated by the polyethylene glycol method [39] with the AMV transcript mixture carrying the citrus leprosis virus C (CiLV-C) MP, citrus leprosis virus C2 (CiLV-C2) MP wild type (MPWT) and the mutated versions
Despite the clear differences observed for cell-to-cell movement, both CiLV-C2 MP variants (MPS72F and MPS259F ) exhibited systemic movement similar to that observed for the control (AMV wild type (WT)), where the presence of viral RNA was detected in all inoculated (I) and upper
Summary
For the cell-to-cell movement assay, a modified infectious AMV cDNA 3 clone (pGFP/. A255/CP) [36], which expresses the green fluorescent protein (GFP), was used to exchange the 255 amino acids (aa) of the AMV MP gene with the corresponding MP of CiLV-C (strain CRD), wild type (WT) CiLV-C2 (strain L147V1) and the mutated versions of CiLVC2 MP where a serine residue was replaced by phenylalanine at position 72 (MPS72F ) or. The genes were amplified from total RNA extracted from infected citrus leaves and fruits or transgenic N. tabacum P12 plants that constitutively express the AMV. Inoculation of P12 Plants and Protoplasts for Analysis of Cell-to-Cell Movement, Systemic. At least three (unless noted otherwise) transgenic N. tabacum P12 plants were grown and inoculated with RNA transcripts, as previously described [38]. Each construct was inoculated on two plants with two (for systemic analysis) or three (for cell-to-cell analysis) leaves per plant, and 15 μL (~3 μg) of the transcription mixture was used as inoculum per leaf. For analysis of AMV RNA accumulation, protoplasts were extracted from P12 leaves and 2.5 × 105 protoplasts were inoculated by the polyethylene glycol method [39] with the AMV transcript mixture carrying the CiLV-C MP, CiLV-C2 MPWT and the mutated versions.
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