Functional Characterization of Replication-Associated Proteins Encoded by Alphasatellites Identified in Yunnan Province, China

Abstract

Alphasatellites, which encode only a replication-associated protein (alpha-Rep), are frequently found to be non-essential satellite components associated with begomovirus/betasatellite complexes, and their presence can modulate disease symptoms and/or viral DNA accumulation during infection. Our previous study has shown that there are three types of alphasatellites associated with begomovirus/betasatellite complexes in Yunnan province in China and they encode three corresponding types of alpha-Rep proteins. However, the biological functions of alpha-Reps remain poorly understood. In this study, we investigated the biological functions of alpha-Reps in post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) using 16c and 16-TGS transgenic Nicotiana benthamiana plants. Results showed that all the three types of alpha-Rep proteins were capable of suppressing the PTGS and reversing the TGS. Among them, the alpha-Rep of Y10DNA1 has the strongest PTGS and TGS suppressor activities. We also found that the alpha-Rep proteins were able to increase the accumulation of their helper virus during coinfection. These results suggest that the alpha-Reps may have a role in overcoming host defense, which provides a possible explanation for the selective advantage provided by the association of alphasatellites with begomovirus/betasatellite complexes.

Keywords: PTGS; TGS; alphasatellite; geminivirus complex; replication-associated protein.

Figure 1

Sequence analysis of alpha-Reps. Amino acid sequence alignment of alpha-Reps of Y10 alphasatellite (AJ579353), Y35 alphasatellite (AJ579345), Y132 alphasatellite (AJ579349), GDarSLA (Gossypium darwinii symptomless alphasatellite, NC_013013), and GMusSLA (Gossypium mustelinium symptomless alphasatellite, NC_013012). Positions with strictly conserved amino acids are highlighted in black, conserved substitutions in dark gray, and blocks of similar residues in light gray.

Figure 2

Alpha-Reps suppress the post-transcriptional gene silencing (PTGS) of local GFP. (A) Schematic diagram showing the constructs used in the local GFP PTGS assay in panel B. The ORFs of Y10alpha-Rep, Y35alpha-Rep, and Y132alpha-Rep were cloned into the binary vector, pCHF3, to be used for transient expression. An empty pCHF3 vector (pCHF3::Vec) was used as the negative control. (B) Suppression of PTGS of GFP in leaves of transgenic N. benthamiana 16c plants. Leaves of 16c plants were coagroinfiltrated with constructs harboring GFP (35S::GFP) and either a pCHF3::Vec, pCHF3::Y10alpha-Rep, pCHF3::Y35alpha-Rep, or pCHF3::Y132alpha-Rep. Leaves of 16c plants coagroinfiltrated with 35S::GFP and TYLCCNV-βC1 or TBSV-P19 were used as the positive controls. The agroinfiltrated leaves were photographed under UV light at 3 and 5 days post infiltration (dpi). (C) Northern blot analysis of GFP mRNA accumulation in agroinfiltrated leaf patches shown in panel B. An ethidium bromide-stained gel shown below the blots provides an RNA-loading control. (D) Western blot (WB) assay of GFP accumulation in agroinfiltrated leaf patches shown in panel B. CBB staining of the large subunit of Rubisco was used as a loading control. For (B–D), the experiments were repeated three times with similar results.

Figure 3

Alpha-Reps suppress the post-transcriptional gene silencing (PTGS) of systemic GFP. GFP fluorescence observed in transgenic N. benthamiana 16c plants agroinoculated with 35S::GFP plus pCHF3::Vec (A) pCHF3::Y10alpha-Rep (B) pCHF3::Y35alpha-Rep (C) pCHF3::Y132alpha-Rep (D) TYLCCNV-βC1 (E) or TBSV-P19 (F) at 30 days post infiltration (dpi). Leaves of 16c plants coagroinfiltrated with 35S::GFP plus pCHF3::Vec were used as the negative control and leaves of 16c plants coagroinfiltrated with 35S::GFP plus TYLCCNV-βC1 or TBSV-P19 were used as the positive controls.

Figure 4

Alpha-Reps suppress the transcriptional gene silencing (TGS) of GFP. (A) Schematic diagram showing the constructs used in the GFP TGS assay in panel B. The ORFs of Y10alpha-Rep, Y35alpha-Rep, and Y132alpha-Rep were cloned into the PVX, to be used for transient expression. An empty PVX vector (PVX::Vec) was used as the negative control. (B) Transgenic N. benthamiana 16-TGS plants were agroinfiltrated with PVX::Y10alpha-Rep, PVX::Y35alpha-Rep, or PVX::Y132alpha-Rep, and the plants were photographed under UV light at 14 dpi. 16-TGS plants agroinoculated with PVX::Vec and PVX::TYLCCNV-βC1 were used as negative and positive controls, respectively. (C) Northern blot analysis of GFP mRNA accumulation in agroinfiltrated leaf patches is shown in panel B. An ethidium bromide-stained gel shown below the blots provides an RNA-loading control. (D) Western blot (WB) assay of GFP accumulation in agroinfiltrated leaf patches shown in panel B. CBB staining of the large subunit of Rubisco was used as a loading control. For (B–D) the experiments were repeated three times with similar results.

Figure 5

Southern blot analysis of viral DNA accumulation in N. benthamiana plants infected by TYLCCNV alone or together with Y10DNA1, Y10alpha-Rep, Y35alpha-Rep, Y132alpha-Rep, or TYLCCNB at 30 dpi. Total genomic DNA (approximately 10 µg for each lane) from a mixture of three seedlings was used for the Southern blot. Blots were probed with the coat protein gene sequence of TYLCCNV (top), the Rep of Y10DNA1 (middle), and the full-length sequence of TYLCCNB (bottom). An ethidium bromide-stained gel shown below the blots provides a DNA-loading control (downmost). The positions of supercoiled (scDNA) and single stranded (ssDNA) forms are indicated, respectively.