Functional Analysis of V2 Protein of Beet Curly Top Iran Virus

Abstract

Geminivirus beet curly top Iran virus (BCTIV) is one of the main causal agents of the beet curly top disease in Iran and the newly established Becurtovirus genus type species. Although the biological features of known becurtoviruses are similar to those of curtoviruses, they only share a limited sequence identity, and no information is available on the function of their viral genes. In this work, we demonstrate that BCTIV V2, as the curtoviral V2, is also a local silencing suppressor in Nicotiana benthamiana and can delay the systemic silencing spreading, although it cannot block the cell-to-cell movement of the silencing signal to adjacent cells. BCTIV V2 shows the same subcellular localization as curtoviral V2, being detected in the nucleus and perinuclear region, and its ectopic expression from a PVX-derived vector also causes the induction of necrotic lesions in N. benthamiana, such as the ones produced during the HR, both at the local and systemic levels. The results from the infection of N. benthamiana with a V2 BCTIV mutant showed that V2 is required for systemic infection, but not for viral replication, in a local infection. Considering all these results, we can conclude that BCTIV V2 is a functional homologue of curtoviral V2 and plays a crucial role in viral pathogenicity and systemic movement.

Keywords: PTGS suppressor; beet curly top disease; hypersensitive response-like; pathogenicity; systemic movement.

Figure 1

(A): Beet curly top Iran virus (BCTIV) genome structure. Arrows represent open reading frames (ORFs). LIR: long intergenic region; SIR: short intergenic region. Common region inside LIR is depicted in black (B): Alignment of the aminoacid sequences of the V2 proteins from the becurtovirus beet curly top Iran virus (BCTIV; AFK14083), the curtovirus beet curly top virus (BCTV; AAA42752.1), the begomoviruses tomato yellow leaf curl virus (TYLCV; CAA33687.1), tomato leaf curl Palampur virus (ToLCPalV; CAP03292), and East African cassava mosaic Cameron virus (EACMCV; AF112354). Gaps (-) were introduced to optimize the alignment. The positions of the predicted putative phosphorylation motifs P1 (protein kinase CK2/protein kinase C), P2 (protein kinase CK2), and P3 (protein kinase C) are depicted in white letters inside black boxes. The hydrophobic domains (H1 and H2) are shadowed in gray.

Figure 2

Local PTGS suppression assay in wild type Nicotiana benthamiana. (A) Leaves co-infiltrated by a mixture of two Agrobacterium tumefaciens cultures expressing GFP and V2 from beet curly Iran top virus (IVV2), under UV light at six days post infiltration (p.i.). V2 from beet curly top virus (BCV2) and the empty vector (EV) were used as controls in (B). Western blot was performed by anti-GFP antibody. Coomassie blue staining of SDS-PAGE gel is shown as a loading control. Four to six plants were agroinfiltrated per experiment. Similar results were obtained in three independent experiments. (C) High-molecular weight Northern blot to detect GFP mRNA accumulation in infiltrated tissues. 10 ug of total RNA was used per lane. The ethidium bromide-stained pattern is shown as a loading control. (D) Small RNA Northern blot analysis of GFP-derived siRNAs (siGFP) in infiltrated tissues was observed. 20 ug of total RNA per lane was observed. Detection of sn U6 is used to control for loading. (E) V2 viral protein transcription was confirmed by RT-PCR, and Elongation factor alpha (NbE1Fa) was used as an internal control.

Figure 3

Effect of V2 from beet curly top Iran virus on long distance and short range spread of RNA silencing in 16c Nicotiana benthamiana plants. (A) 16c plants were agroinfiltrated with two A. tumefaciens cultures expressing GFP and V2 from beet curly top Iran virus (IVV2). V2 from beet curly top virus (BCV2), P19, and the empty vector (EV) were used as controls. Agroinfiltrated 16 c plants were observed under UV light at 18 days post inoculation (dpi). (B) Levels of systemic silencing (silencing index) of these plants at 8-, 18-, and 30-days post infiltration (dpi). Silencing index ranges from 0 (no silenced leaves) to 5 (plant with all the leaves silenced). Values correspond to the average of six plants. Bars represent standard deviation. (C) GFP expression in the cells surrounding the agroinfiltrated area at 6 dpi. Barr: 2 mm. Four to six plants were agroinfiltrated per experiment. Similar results were obtained in three independent experiments.

Figure 4

Local and systemic symptoms induced by Potato virus X (PVX) expression of V2 from beet curly top Iran virus (PVX-IVV2) in wt Nicotiana benthamiana plants. Empty PVX vector (PVX) was used as negative control. Recombinant PVX viruses expressing either the nonstructural protein (NSs) from tomato spotted wilt virus (PVX-NSs) or the V2 protein from beet curly top virus (PVX-BCV2) were used as positive controls. (A) Local symptoms induced in the infiltrated area at 6 dpi. (B) Representative plants showing systemic symptoms at 16 dpi. Four to six plants were agroinfiltrated per experiment. Similar results were obtained in three independent experiments.

Figure 5

Subcellular localization of V2 from beet curly top Iran virus fused to GFP in epidermal cells of wild type Nicotiana benthamiana. (A) Leaves were agroinfiltrated with a construct expressing the GFP (GFP), GFP-V2 fusion protein from beet curly top Iran virus (GFP-IVV2), or the GFP-V2 fusion protein from beet curly top virus (GFP-BCV2). Samples were observed under the confocal microscope at 36 h post infection. (B) Close up confocal images of the observed areas. GFP fluorescence (GFP), autofluorescence (AF), and the bright field channel (BF) are shown.

Figure 6

Infection of wild type Nicotiana benthamaiana plants with wild type beet curly top Iran virus and the V2 stop mutant virus. Plants were agroinoculated with wild-type (wt BCTIV) or V2 mutant BCTIV (BCTIV V2stp) infectious clones. (A) Representative plants showing BCTIV symptoms at 28 dpi. Relative viral DNA accumulation in apical leaves (systemic infection) at 28 dpi (B) or in the infiltrated leaves (local infection) at 6 dpi (C). BCTIV accumulation was measured by qPCR after DpnI treatment to remove bacterial DNA (6 dpi). The same DpnI treatment was applied on bacterial plasmids containing the BCTIV infectious clone as a control (Plasmid). DNA levels were normalized to Elongation factor alpha (NbE1Fa) and are presented as the relative amount of virus compared with the amount found in wild-type BCTIV samples (set to 1). Bars represent mean values ± standard error (SE) for six pools of two leaves. Mean values marked with different letters (a or b) indicate results significantly different from each other, as established by Student’s t-test α < 0.001) virus titer.