A maize triacylglycerol lipase inhibits sugarcane mosaic virus infection

Abstract

Triacylglycerol lipase (TGL) plays critical roles in providing energy for seed germination and plant development. However, the role of TGL in regulating plant virus infection is largely unknown. In this study, we adopted affinity purification coupled with mass spectrometry and identified that a maize (Zea mays) pathogenesis-related lipase protein Z. mays TGL (ZmTGL) interacted with helper component-proteinase (HC-Pro) of sugarcane mosaic virus (SCMV). Yeast two-hybrid, luciferase complementation imaging, and bimolecular fluorescence complementation assays confirmed that ZmTGL directly interacted with SCMV HC-Pro in vitro and in vivo. The 101-460 residues of SCMV HC-Pro were important for its interaction with ZmTGL. ZmTGL and SCMV HC-Pro co-localized at the mitochondria. Silencing of ZmTGL facilitated SCMV infection, and over-expression of ZmTGL reduced the RNA silencing suppression activity, most likely through reducing HC-Pro accumulation. Our results provided evidence that the lipase hydrolase activity of ZmTGL was associated with reducing HC-Pro accumulation, activation of salicylic acid (SA)-mediated defense response, and inhibition of SCMV infection. We show that ZmTGL inhibits SCMV infection by reducing HC-Pro accumulation and activating the SA pathway.

Figure 1

Purification and identification of maize partners interacting with SCMV HC-Pro. A, Schematic representation of pSCMV-Strep-HC-Pro infectious clone. The Strep-tag encoding sequence was marked in red at the 5′-terminus of HC-Pro in pSCMV vector. SCMV protein: P1: the first protein; P3: the third protein; 6K1: the first 6K protein; CI: cytoplasmic inclusion protein, 6K2: the second 6K protein, VPg: viral genome-linked protein, NIa: nuclear inclusion protein a-proteinase, NIb: nuclear inclusion protein b, CP: coat protein, P3N-PIPO: the N-terminal half of P3 fused to Pretty Interesting Potyvirus Open Reading Frame. B, Typical mosaic symptoms on maize inbred lines B73 plants infected with wild-type SCMV (left) and SCMV-Strep-HC-Pro (right), respectively, at 10 dpi. C, SDS and western blotting analyses of the proteins purified from the maize leaves infected with SCMV and SCMV-Strep-HC-Pro, respectively. D, Peptides of ZmTGL (GenBank accession: NP 001149280) were identified in SCMV-Strep-HC-Pro-purified samples through LC–MS/MS and shown in red.

Figure 2

Knockdown of ZmTGL in maize plants increased the accumulation level of SCMV. A, Phenotype of maize plants infected with FoMV-sg or FoMV-ZmTGL at 12 dpi. B, qRT-PCR analysis of the relative accumulation levels of ZmTGL mRNA in the second upper leaves systemically infected by FoMV-sg and FoMV-ZmTGL at 12 dpi. The bar graphs represented the means ± standard deviations of three biological replicates and three technical replicates. Statistically significant differences between means were determined by employing Tukey multiple range test for between-group comparisons. Different letters indicated significant differences (P < 0.05). C, Phenotypes of maize plants was photographed at 6 d postchallenge inoculation with SCMV-GFP under daylight and UV light. D, qRT-PCR analysis of the relative accumulation levels of SCMV RNA in the SCMV-GFP first systemically infected leaves at 6 dpi. The bar graphs represented the means ± standard deviations of three biological replicates and three technical replicates. Statistically significant differences between means were determined by employing Tukey multiple range test for between-group comparisons. Different letters indicated significant differences (P < 0.05). E, Western blotting analysis of SCMV CP and HC-Pro accumulation levels in the SCMV-GFP first systemically infected leaves at 6 dpi. Lower panel, PSS as a loading control. Band intensities were measured using the ImageJ software. Numbers indicate the accumulation levels of SCMV CP and HC-Pro normalized to PSS staining. Three independent experiments are conducted.

Figure 3

ZmTGL interacted with SCMV HC-Pro in vitro and in vivo. A, Y2H analysis of the interaction between SCMV HC-Pro and ZmTGL in vivo. The transformed yeast cells were subjected to 10-fold serial dilutions and plated on SD-LWHA medium for 4 d. Yeast cells co-transformed with AD-HC-Pro and BD-HC-Pro were served as positive controls; yeast cells co-transformed with AD-T7 and BD-lam, AD and BD-HC-Pro, or AD and BD-ZmTGL were used as negative controls. On the left, the schematic representation indicated the positioning of each plasmid combination. B, LCI analysis of the interaction between ZmTGL and SCMV HC-Pro in N. benthamiana leaves. NLuc co-expressed with CLuc or CLuc-HC-Pro-Pro, and ZmTGL-NLuc co-expressed with CLuc, were used as negative controls. The luciferase activity was measured using a luminometer at 48 hpai. The bar graphs represented the means ± standard deviations of three biological replicates. Statistically significant differences between means were determined by employing Tukey multiple range test for between-group comparisons. Different letters indicated significant differences (P < 0.05). C, BiFC analysis of the interaction between ZmTGL and HC-Pro in N. benthamiana leaves. Co-expression of YN and ZmTGL-YC or YN-HC-Pro and YC were used as negative controls. Confocal imaging was performed at 48 hpai. Bars = 10 μm.

Figure 4

ZmTGL/HC-Pro interaction complex localized to mitochondria. A, Confocal micrographs of N. benthamiana leave cells co-expressing GFP and ZmTGL-DsRed, GFP-HC-Pro, and DsRed, or GFP-HC-Pro and ZmTGL-DsRed at 48 hpai. Images (left to right) showed GFP fluorescence, DsRed fluorescence, and overlay of the two images. White arrows indicated co-localization of GFP-HC-Pro and ZmTGL-DsRed. Bars = 10 μm. B, ZmTGL-GFP and ZmTGL/HC-Pro interaction complex co-localized with ScCOX4-DsRed (mitochondria marker), but GFP-HC-Pro alone did not co-localize with ScCOX4-DsRed in N. benthamiana leaf cells at 48 hpai. White arrows indicate co-localization of the mitochondria marker ScCOX4-DsRed with ZmTGL-GFP or ZmTGL-HC-Pro-Pro interaction complex. Bars = 10 μm.

Figure 5

Over-expression of ZmTGL reduced the RNA silencing suppression activity and accumulation of SCMV HC-Pro. A, GFP-expressing N. benthamiana (16C) leaves were infiltrated with a mixture of Agrobacterium cultures carrying different constructs and photographed at 6 dpai. The leave patches expressing GFP and GUSp-Myc were used as negative controls. The images represented at least 12 individual leaf samples. B, qRT-PCR analysis of GFP mRNA accumulation levels in agroinfiltrated 16C leaf patches at 6 dpai. The bar graphs represented the means ± standard deviations of three biological replicates and three technical replicates. Statistically significant differences between means were determined by employing Tukey multiple range test for between-group comparisons. Different letters indicated significant differences (P < 0.05). C, Western blotting analysis of GFP, HC-Pro, GUSp-Myc, and ZmTGL-Myc accumulation levels in the 16C leaf patches at 6 dpai. Numbers indicate the relative accumulation levels of GFP and HC-Pro normalized to PSS staining. D, N. benthamiana leaves were infiltrated with a mixture of Agrobacterium cultures carrying different constructs and photographed at 3 dpai. The images represented at least 10 individual leaf samples. E, qRT-PCR analysis of GFP mRNA accumulation levels in agroinfiltrated N. benthamiana leaf patches at 3 dpai. The bar graphs represented the means ± standard deviations of three biological replicates and three technical replicates. Statistically significant differences between means were determined by employing Tukey multiple range test for between-group comparisons. Different letters indicated significant differences (P < 0.05). F, Western blotting analysis of GFP, HC-Pro, GUSp-Myc, and ZmTGL-Myc accumulation levels in agroinfiltrated leaf patches at 3 dpai. Numbers indicate the relative accumulation levels of GFP and HC-Pro normalized to PSS staining. G, DsRed-fused ZmTGL had no effect on GFP expression. H, ZmTGL-DsRed reduced GFP-fused HC-Pro protein accumulation. The pictures in G and H were taken at 3 dpai. Red asterisks indicated ZmTGL-DsRed. I, GFP fluorescence in local patches co-expressing GFP-HC-Pro (OD₆₀₀ = 0.5) and ZmTGL-DsRed (OD₆₀₀ = 0, 0.1, 0.3, or 0.5). Western blotting analysis of GFP-HC-Pro and ZmTGL-DsRed accumulation levels in agroinfiltrated leaf patches. Numbers indicate the relative accumulation levels of GFP-HC-Pro and ZmTGL-DsRed normalized to PSS staining. The experiments are repeated thrice independently.

Figure 6

Role of lipase hydrolase activity of ZmTGL in the accumulation levels and RNA silencing suppression activity of SCMV HC-Pro. A, Analysis of GFP-HC-Pro accumulation in N. benthamiana leaves co-expressing with wild-type ZmTGL-DsRed or its hydrolase activity-defective mutant ZmTGL-S161S/D255S/H304A-DsRed mutant at 3 dpai. Numbers indicate the relative accumulation levels of GFP-HC-Pro, ZmTGL-DsRed, and its mutant normalized to PSS staining. B, Y2H analysis of the interaction of SCMV HC-Pro with wide-type ZmTGL or ZmTGL-S161A/D255A/H304A mutant. C, The HC-Pro RNA silencing suppression activity co-expressing with wide-type ZmTGL or ZmTGL-S161S/D255S/H304A mutant in RNA silencing suppression assay. The leave patches co-expressing GFP and GUSp-Myc were used as negative controls. The images represented at least 10 individual leaf samples. D, qRT-PCR analysis of GFP mRNA accumulation levels in agroinfiltrated 16C leaf patches at 6 dpai. The bar graphs represented the means ± standard deviations of three biological replicates and three technical replicates. Statistically significant differences between means were determined by employing Tukey multiple range test for between-group comparisons. Different letters indicated significant differences (P <  0.05). E, Western blotting analysis of GFP, HC-Pro, GUSp-Myc, and ZmTGL-Myc accumulation levels in the leaf patches at 6 dpai. Numbers indicate the relative accumulation levels of GFP and HC-Pro normalized to PSS staining. The experiments are repeated thrice independently.

Figure 7

ZmTGL over-expression enhanced SA-mediated defense response and SA treatment inhibited SCMV infection in maize plants. A, Concentrations of SA in maize plants inoculated with pV101-GUSp-Myc, pV101-ZmTGL-Myc, and pV101-ZmTGL-S161A/D255A/H304A-Myc at 8 dpi, respectively. The bar graphs represented the means ± standard deviations of three replicates. Statistically significant differences between means were determined by employing Tukey multiple range test for between-group comparisons. Different letters indicated significant differences (P  < 0.05). B, qRT-PCR analysis of SA marker genes ZmPR3, ZmPR4, and ZmPR5 mRNA accumulation levels in the first systemically leaves of maize plants inoculated with pV101-GUSp-Myc, pV101-ZmTGL-Myc, and pV101-ZmTGL-S161A/D255A/H304A-Myc at 8 dpi. The bar graphs represented the means ± standard deviations of three biological replicates and three technical replicates. Statistically significant differences between means were determined by employing two-tailed Student’s t test. The asterisks represent statistical significance (^*P < 0.05; ^**P < 0.01). C, Symptoms of maize plants infected with SCMV-GFP after the pretreatment with SA or H₂O at 6 dpi. D, Western blotting analysis of SCMV accumulation levels in the maize leaves infected with SCMV-GFP at 6 dpi. Numbers indicate the relative accumulation levels of SCMV CP normalized to PSS staining. The experiments are repeated thrice independently.

Figure 8

Effect of mutation on the hydrolase active sites of ZmTGL on SCMV accumulation in maize plants. A, Phenotypes of the maize plants individually infected with SCMV-GUSp-Myc, SCMV-ZmTGL-Myc, or SCMV-ZmTGL-S161A/D255A/H304A-Myc. The pictures were taken at 8 dpi under daylight. B, Western blotting analysis of SCMV CP and HC-Pro accumulation levels in the maize leaves individually infected with SCMV-GUSp-Myc, SCMV-ZmTGL-Myc, or SCMV-ZmTGL-S161A/D255A/H304A-Myc at 8 dpi. Numbers indicate the accumulation levels of SCMV CP and HC-Pro normalized to PSS staining. The experiments are repeated thrice independently.

Figure 9

Proposed model for the roles of ZmTGL in inhibiting SCMV infection. HC-Pro, the viral RSS, can counteract host RNA silencing immune response and facilitate SCMV infection in maize plants. ZmTGL interacts with SCMV HC-Pro and disrupts HC-Pro accumulation via the hydrolase activity of ZmTGL to inhibit SCMV infection. SCMV infection induces ZmTGL upexpression in maize plants, which stimulates SA accumulation to activate the SA-mediated defense response.