Chinese wheat mosaic virus-derived vsiRNA-20 can regulate virus infection in wheat through inhibition of vacuolar- (H+ )-PPase induced cell death

Abstract

Vacuolar (H⁺ )-PPases (VPs), are key regulators of active proton (H⁺ ) transport across membranes using the energy generated from PPi hydrolysis. The VPs also play vital roles in plant responses to various abiotic stresses. Their functions in plant responses to pathogen infections are unknown. Here, we show that TaVP, a VP of wheat (Triticum aestivum) is important for wheat resistance to Chinese wheat mosaic virus (CWMV) infection. Furthermore, overexpression of TaVP in plants induces the activity of PPi hydrolysis, leading to plants cell death. A virus-derived small interfering RNA (vsiRNA-20) generated from CWMV RNA1 can regulate the mRNA accumulation of TaVP in wheat. The accumulation of vsiRNA-20 can suppress cell death induced by TaVP in a dosage-dependent manner. Moreover, we show that the accumulation of vsiRNA-20 can affect PPi hydrolysis and the concentration of H⁺ in CWMV-infected wheat cells to create a more favorable cellular environment for CWMV replication. We propose that vsiRNA-20 regulates TaVP expression to prevent cell death and to maintain a weak alkaline environment in cytoplasm to enhance CWMV infection in wheat. This finding may be used as a novel strategy to minimize virus pathogenicity and to develop new antiviral stratagems.

Keywords: Chinese wheat mosaic virus (CWMV); pathogenicity; vacuolar (H+)-PPase (VP); virus-derived small interfering RNA (vsiRNA); wheat.

Figure 1

Sequence alignment using vacuolar (H⁺)‐PPase protein sequences from different plant species. (a) A phylogenetic tree constructed using the Triticum aestivum vacuolar (H⁺)‐PPase (TaVP) sequence and its closely related vacuolar (H⁺)‐PPase protein sequences from other plant species. TaVP is marked by a red box. The GenBank accession number of each protein is shown. The names of plant species are presented as: Sorghum bicolor (Sb), Zea mays (Zm), Setaria italica (Si), Panicum hallii (Ph), Brachypodium distachyon (Bd), Hordeum vulgare (Hv), T. aestivum (Ta), Oryza sativa (Os), Arabidopsis thaliana (At), Brassica napus (Bn), Gossypium raimondii (Gr), Solanum lycopersicum (Sl), Vitis vinifera (Vv), Nicotiana attenuata (Na), Sorghum bicolor (Sb), Glycine max (Gm). (b) Subcellular localization of TaVP protein. TaVP protein fused to green fluorescent protein (TaVP‐GFP) and O. sativa vacuolar Pi efflux transporters 2 protein fused to red fluorescent protein (OsVPE2‐RFP) were co‐expressed in wheat protoplasts under the control of the Cauliflower mosaic virus (CaMV) 35S promoter and observed under a confocal microscope. The photographs were taken in bright light for cell morphology, in dark field for green or red fluorescence, and in combination for yellow fluorescence. Bars, 50 µm.

Figure 2

Expression of Triticum aestivum vacuolar (H⁺)‐PPase (TaVP) and accumulations of Chinese wheat mosaic virus (CWMV) in wheat at different numbers of d post‐inoculation (dpi) with virus. Expression of TaVP and accumulations of CWMV in CWMV‐inoculated wheat plants were determined by quantitative reverse transcription (qRT‐PCR) using TaVP or CWMV coat protein (CP) gene‐specific primers. The relative expression levels of TaVP or CWMV CP gene were calculated using the 2^{−△△C T} method. The expression level of the TaActin gene was used as an internal control. Each relative expression level is presented as mean ± SD from three biological samples and each biological sample had three technical replicates. Statistical analyses were done using Student's t‐test. *, P < 0.05; **, P < 0.01; ns, no significant difference.

Figure 3

Effect of Triticum aestivum vacuolar (H⁺)‐PPase (TaVP) on Chinese wheat mosaic virus (CWMV) genomic RNA (gRNA) accumulation in wheat. (a) Reverse transcription PCR (RT‐PCR) detection of Barley stripe mosaic virus (BSMV) and CWMV infections in the BSMV + CWMV (i) or BSMV:TaVP + CWMV (ii) co‐inoculated wheat plants using BSMV RNAγ or CWMV RNA2 specific primers. The RT‐PCR products were visualized in agarose gels. Ten plants were analyzed for each treatment. Total RNA from a mock‐inoculated wheat plant was used as a negative control (−). Diluted plasmid pCB‐γ and pCB‐T7‐R2 were used as the positive control (+) for BSMV and CWMV, respectively. (b) Relative expression levels of TaVP in eight wheat plants co‐infected with CWMV and BSMV:TaVP. Total RNA from a BSMV and CWMV co‐infected wheat plant was used as a control. Each relative expression level is presented as mean ± SD from three biological samples and each biological sample had three technical replicates. Statistical analyses were done using Student's t‐test. *, P < 0.05; **, P < 0.01; ns, no significant difference. (c) Phenotypes in the fourth leaves of the plants inoculated with phosphate buffered saline (PBS) as Mock, CWMV, BSMV, BSMV:PDS, BSMV:TaVP, BSMV + CWMV and BSMV:TaVP + CWMV, respectively. Photographs were taken at 14 d post‐inoculation (dpi) with virus. (d) Detection of CWMV gRNAs in the fourth leaves of the plants co‐inoculated with BSMV + CWMV or BSMV:TaVP + CWMV through a Northern blot assay at 14 dpi. The ethidium bromide stained gel was used to show RNA loadings. Each treatment had three plants. (e) Expression of TaVP in the agro‐infiltrated N. benthamiana leaves were determined by Western blot using an HA‐specific antibody. Coomassie brilliant blue (CBB) stained rubisco gel was used to show the protein loadings. (f) Detection of CWMV gRNAs in the leaves co‐infiltrated with p35S:TaVP‐HA and the CWMV infectious clone or p35S:00 and the CWMV infectious clone through Northern blot at 4 dpi. The ethidium bromide stained gel is used to show RNA loadings. Three plants were used to represent a treatment.

Figure 4

Overexpression of Triticum aestivum vacuolar (H⁺)‐PPase (TaVP) increases pyrophosphate (PPi) hydrolysis, leading to plant cell death. (a) Substrate hydrolysis by the vacuolar (H⁺)‐PPases (VPs) in crude membrane extracts from plants inoculated with 35S:00 or 35S:TaVP‐HA at 5 d post‐inoculation (dpi) with virus. Crude membrane extracts were prepared from leaves of > 10 plants and used for enzyme activity assays as described in the Materials and Methods section. Each result (mean ± SD) was from three biological samples and each biological sample had three technical replicates. Statistical analyses were done using Student's t‐test. **, P < 0.01. (b) At 5 dpi, the 35S:00 or 35S:TaVP‐HA‐infiltrated Nicotiana benthamiana leaves were detached and analyze for cell death through Trypan blue staining. The experiment was repeated at least three times with five or more plants per treatment.

Figure 5

Targeting the 3′‐ untranslated region (UTR) of Triticum aestivum vacuolar (H⁺)‐PPase (TaVP) by virus‐derived small interfering RNA‐20 (vsiRNA‐20). (a) A schematic diagram showing the base pairing between vsiRNA‐20 and TaVP. The noncapped 5′ remnants of TaVP generated by vsiRNA‐20 cleavage were determined by 5′ rapid amplification of cDNA ends (5′‐RACE) using 30 selected clones. The frequencies of clones showing noncapped 5′ remnants are indicated. The predicted cleavage site is in red and the additional cleavage site is in black and bold. (b) Northern blot analysis of vsiRNA‐20 generated from Chinese wheat mosaic virus (CWMV) genome. Total RNA was extracted from three CWMV‐infected and three mock‐inoculated wheat plants. Expression of U6 was used as an internal control. (c) Schematic diagrams showing the structures of p35S:GFP, p35S:vsiRNA‐20, p35S:vsiRNA‐12 and p35S:GFP‐UTR, respectively. (d) Green fluorescent protein (GFP) fluorescence in Nicotiana benthamiana leaves co‐infiltrated with p35S:GFP + p35S:vsiRNA‐20, p35S:GFP‐UTR + p35S:vsiRNA‐20, p35S:GFP + p35S:vsiRNA‐12 or p35S:GFP‐UTR + p35S:vsiRNA‐12. The leaves were photographed under an UV light at 4 d post‐inoculation (dpi) with virus. (e) Detection of vsiRNA‐20 or vsiRNA‐12 accumulation in N. benthamiana leaves co‐infiltrated with (1) p35S:GFP + p35S:vsiRNA‐12, (2) p35S:GFP‐UTR + p35S:vsiRNA‐12, (3) p35S:GFP + p35S:vsiRNA‐20, (4) p35S:GFP‐UTR + p35S:vsiRNA‐20, (5) p35S:GFP + p35S:vsiRNA‐20, (6) p35S:GFP‐UTR + p35S:vsiRNA‐20, (7) p35S:GFP + p35S:vsiRNA‐12, and (8) p35S:GFP‐UTR + p35S:vsiRNA‐12 through Northern blot assays. Expression of U6 was used as an internal control. This experiment was repeated three times. (f) Detection of GFP in the N. benthamiana leaves co‐infiltrated with (1) p35S:GFP + p35S:vsiRNA‐20, (2) p35S:GFP + p35S:vsiRNA‐12, (3) p35S:GFP‐UTR + p35S:vsiRNA‐12, and (4) p35S:GFP‐UTR + p35S:vsiRNA‐20. Coomassie brilliant blue (CBB) large stained rubisco gel was used to show protein loadings. (g) Relative expression levels of GFP in N. benthamiana leaves co‐infiltrated with p35S:GFP + p35S:vsiRNA‐20, p35S:GFP + p35S:vsiRNA‐12, p35S:GFP‐UTR + p35S:vsiRNA‐12, and p35S:GFP‐UTR + p35S:vsiRNA‐20. Total RNA from a p35S:GFP + p35S:vsiRNA‐20 co‐infected leaves was used as a control. Each relative expression level is presented as mean ± SD from three biological samples and each biological sample had three technical replicates. Statistical analyses were done using Student's t‐test. **, P < 0.01; ns, no significant difference.

Figure 6

Influence of virus‐derived small interfering RNA‐20 (vsiRNA‐20) on Chinese wheat mosaic virus (CWMV) gRNA accumulations in wheat. (a) Schematic diagrams showing the construction of the pCB‐T7‐R1M mutant from plasmid pCB‐T7‐R1. (b) Phenotypes of wheat plants inoculated with phosphate buffered saline (PBS) buffer as wild‐type (WT), CWMV or its mutant (CWMVm). The plants were photographed at 98 d post‐inoculation (dpi) with virus. (c) Detection of vsiRNA‐20 accumulation in systemic leaves of wheat plants inoculated with CWMV or CWMVm transcripts, or with PBS buffer as WT through Northern blot assays. The expression of U6 was used as an internal control. The experiment was repeated three times. (d) Detection of CWMV gRNAs in wheat plants inoculated with CWMV or CWMVm transcripts through Northern blot at 98 dpi. The ethidium bromide stained gel was used to show RNA loadings. (e) The expression level of TaVP in wheat plants inoculated with CWMV or CWMVm transcripts, or with PBS buffer (WT). This experiment was repeated three times. Standard errors of individual means are shown. Statistical analyses were done using Student's t‐test. **, P < 0.01; ns, no significant difference. (f) A schematic diagram showing the base pairing between vsiRNA‐20 and TaVP in wheat inoculated with CWMV. The noncapped 5′ remnants of TaVP generated by vsiRNA‐20 cleavage was determined by 5′ rapid amplification of cDNA ends (5′‐RACE) using 30 selected clones. The frequencies of clones showing noncapped 5′ remnants are indicated. The predicted cleavage site is in red.

Figure 7

The vacuolar (H⁺)‐PPases (VPs) and the vacuolar (H⁺)‐ATPase (V‐ATPase) activities, and intracellular pH values in wheat protoplasts transfected with Chinese wheat mosaic virus (CWMV) or its mutant (CWMVm). Substrate hydrolysis activity of VP (a) and V‐ATPase (b) in crude membrane extracts from plants inoculated with buffer (Mock), CWMV or CWMVm at 10 and 30 d post‐inoculation (dpi) with virus, respectively. Crude membrane extracts were prepared from > 30 plants and used for enzyme activity assays. (c) Intracellular pH values in wheat protoplasts transfected with buffer (Mock), CWMV or CWMVm transcripts determined using BCECF‐AM dye (n = 10). The fluorescence ratios are shown in pseudocolor images and the emission at the corresponding excitation wavelength is presented in grayscale. (d) The relative transcripts level of viral RNA in protoplasts inoculated with CWMV or CWMVm. Each relative transcripts level is presented as mean ± SD from three biological samples and each biological sample had three technical replicates. Statistical analyses were done using Student's t‐test. **, P < 0.01; ns, no significant difference.

Figure 8

The virus‐derived small interfering RNA‐20 (vsiRNA‐20) can suppress cell death in Nicotiana benthamiana leaves overexpressing Triticum aestivum vacuolar (H⁺)‐PPase (TaVP). (a) At 5 d post‐inoculation (dpi) with virus, N. benthamiana leaves infiltrated with 35S:TaVP‐HA, 35S:00, 35S:TaVP‐HA + 35S:vsiRNA‐20 or 35S:TaVP‐HA + 35S:vsiRNA‐12 were detached, photographed and analyzed for cell death using Trypan blue staining. The experiment was repeated at least three times, using five or more plants per treatment. (b) Detection of vsiRNA accumulation by Northern blot assay. Total RNA was extracted from N. benthamiana leaves infiltrated with 35S:TaVP‐HA, 35S:TaVP‐HA + 35S:vsiRNA‐20 or 35S:TaVP‐HA + 35S:vsiRNA‐12 at 5 dpi. The resulting RNA samples were used for Northern blot assays. This experiment was repeated at least three times. (c) detection of TaVP‐HA in the N. benthamiana leaves infiltrated with 35S:TaVP‐HA, 35S:TaVP‐HA + 35S:vsiRNA‐20 or 35S:TaVP‐HA + 35S:vsiRNA‐12 through Western blot assays at 5 dpi. The Coomassie brilliant blue (CBB) stained large rubisco gel was used to show protein loadings. This experiment was repeated at least three times. (d) At 5 dpi, N. benthamiana leaves co‐infiltrated with 35S:TaVP‐HA and 35S:vsiRNA‐20 were detached, photographed and analyzed for cell death using Trypan blue staining. The concentration ratio (OD₆₀₀) of 35S:TaVP‐HA/ 35S:vsiRNA‐20 was 1 : 0.5, 1 : 1, 1 : 2, 1 : 4, 1 : 6 and 1 : 8, respectively. The experiment was repeated at least three times, using five or more plants per treatment.

Figure 9

Chinese wheat mosaic virus (CWMV)‐infected wheat leaves were analyzed for cell death through Trypan blue staining. At 20 d post‐inoculation (dpi) with virus, wheat leaves inoculated with Barley stripe mosaic virus (BSMV:00), BSMV:TaVP, CWMV and its mutant (CWMVm) were detached, photographed and analyzed for cell death using Trypan blue staining. The experiment was repeated at least three times, using ≥ 10 plants per treatment.

Figure 10

A working model for virus‐derived small interfering RNA‐20 (vsiRNA‐20) function in Chinese wheat mosaic virus (CWMV) infection in wheat. (I) An uninfected wheat cell. (II) A wheat cell at the early stage of CWMV infection. The CWMV infection upregulated the expression of Triticum aestivum vacuolar (H⁺)‐PPase (TaVP). The increased level of TaVP rapidly increased the number of operating pumps, changed the intracellular pH values, and altered the chemiosmotically derived cation/H⁺ exchange through membrane, leading to an acidified cytoplasm and cell death. This cell death restricts CWMV infection in localized areas. (III) As CWMV infection progresses, abundant vsiRNA‐20 will accumulate in the cytosol. The accumulated vsiRNA‐20 will target TaVP expression in cells to decrease the level of TaVP in tonoplast, leading a change of intracellular pH values and then alterations of chemiosmotically driven cation/H⁺ exchange through membrane. These changes will result in a weak alkalization in the cytosol to prevent cell death and promote CWMV accumulation in cells. Red, upregulated expression; green, downregulated expression.