Viral Perturbation of Alternative Splicing of a Host Transcript Benefits Infection

Abstract

Pathogens disturb alternative splicing patterns of infected eukaryotic hosts. However, in plants it is unknown if this is incidental to infection or represents a pathogen-induced remodeling of host gene expression needed to support infection. Here, we compared changes in transcription and protein accumulation with changes in transcript splicing patterns in maize (Zea mays) infected with the globally important pathogen sugarcane mosaic virus (SCMV). Our results suggested that changes in alternative splicing play a major role in determining virus-induced proteomic changes. Focusing on maize phytoene synthase1 (ZmPSY1), which encodes the key regulatory enzyme in carotenoid biosynthesis, we found that although SCMV infection decreases total ZmPSY1 transcript accumulation, the proportion of splice variant T001 increases by later infection stages so that ZmPSY1 protein levels are maintained. We determined that ZmPSY1 has two leaf-specific transcripts, T001 and T003, distinguished by differences between the respective 3'-untranslated regions (UTRs). The shorter 3'-UTR of T001 makes it the more efficient mRNA. Nonsense ZmPSY1 mutants or virus-induced silencing of ZmPSY1 expression suppressed SCMV accumulation, attenuated symptoms, and decreased chloroplast damage. Thus, ZmPSY1 acts as a proviral host factor that is required for virus accumulation and pathogenesis. Taken together, our findings reveal that SCMV infection-modulated alternative splicing ensures that ZmPSY1 synthesis is sustained during infection, which supports efficient virus infection.

Figure 1.

Development of systemic symptoms in maize seedlings infected with SCMV. A, Infection progress and mosaic/streaking symptoms on the first and second systemically infected leaves of SCMV-inoculated maize seedlings and equivalent leaves of mock-inoculated plants (Mock) at 3, 5, 7, and 9 dpi. The white dashed boxes indicated regions showing mosaic/streaking symptoms as they expanded between 5 and 9 dpi. Scale bars = 0.5 cm. B, Levels of photosynthetic pigments in the first systemically infected leaves over the course of infection. Data were presented as means ± se (n = 3 to 5). Asterisks indicate the significant differences between mock-inoculated and SCMV-infected plants as evaluated by two-tailed Student’s t tests. C, Accumulation of SCMV RNA and CP levels in the first systemically infected leaves of mock-inoculated and SCMV-infected maize seedlings at 3 and 9 dpi. RT-qPCR data (top) are presented as means ± se (n = 4) and relative to ZmUbi (ubiquitin). Asterisks indicate significant differences between 3 and 9 dpi in SCMV-infected plants determined by the two-tailed Student’s t test. Immunoblots (bottom) show SCMV CP (arrowhead) accumulation at 3 and 9 dpi. Actin was used as a loading control.

Figure 2.

Workflow and global analysis of DEGs and DEPs in maize seedlings responding to SCMV infection at the presymptomatic (3 dpi) and steady-symptom (9 dpi) stages of systemic infection. A, Workflow for the integrated transcriptomic and proteomic analyses. Images of aboveground tissues from representative mock-inoculated or SCMV-infected maize seedlings at 3 and 9 dpi are shown. Maize aboveground tissues (n = 3) were harvested, pooled, and used to extract total RNA. RNA-seq allowed identification of DEGs and differentially spliced transcripts. The same aboveground samples were subjected to iTRAQ labeling (n = 2), followed by differential expression analysis. Changes in expression at the post-transcriptional level were assessed by cross-correlating transcriptomic and proteomic data with alternative splicing analysis, followed by biological function analysis of the ZmPSY1 transcripts that responded to SCMV infection. B, The Venn diagram indicates overlap between DEGs affected by only x₁ (item of Infection Status) itself and DEGs affected by x₁ × x₂ (interaction item of Infection Status and Infection Stage) represented in a green circle and a blue circle, respectively. Numbers and percentages of DEGs 1, 2, and 3 are shown. DEG 1 did not change with x₁ × x₂ and contained two types shown in pink-colored schematic line graphs below. These colored schematic line graphs (“fictional models”) were used as a classification method. The y-axes of the line graphs represent gene expression level (TPMs). Asterisks indicate significant differences between mock-inoculated and SCMV-infected samples at 3 or 9 dpi. The DEG 1 was affected only by x₁ and contained 413 upregulated DEGs and 1,296 downregulated DEGs. The DEG 2 was affected only by x₁ × x₂, and contained two types at 9 dpi (93 upregulated and 1,148 downregulated DEGs). The DEG 3 was affected by both x₁ and x₁ × x₂, and contained two types (5,627 upregulated and 1,557 downregulated genes) at 3 dpi. The degree of difference for DEG 3 was variable at 9 dpi due to x₁ × x₂ effect. C, Venn diagrams showing the overlaps between the upregulated (left) and downregulated (right) DEPs at 3 and 9 dpi.

Figure 3.

SCMV infection modifies the accumulation dynamics of ZmPSY1 transcripts but maintains ZmPSY1 synthesis. A, Differential counting bin usages identified ZmPSY1 as a gene giving rise to differentially alternatively spliced transcripts. From this maize genome annotation (http://plants.ensembl.org/Zea_mays/Info/Index), ZmPSY1 has two annotated transcripts, T001 and T002, involving seven exons (gray rectangles) with the sixth having alternative boundaries. Counting bins (shaded boxes) were constructed as depicted, and the sixth exon of variable length was split into two bins (counting bin E006 and E007). The arrowheads indicate alternative splicing sites. The y axis represented normalized read counts from RNA-seq in corresponding counting bins. The counting bin E007 that exhibited significantly differential usage between SCMV-infected and mock-inoculated (Mock) is indicated by a magenta dashed vertical line. Data of E007 shown in the larger magenta dashed box were higher magnification of the boxed area with a magenta dashed vertical line. Data for four treatments, including 3-dpi mock, 3-dpi SCMV, 9-dpi mock, and 9-dpi SCMV, are shown as red, green, blue, and purple lines, respectively. Schematic organizations of alternatively spliced ZmPSY1 transcripts are shown with the coding sequences in red rectangles and the UTRs in black boxes. B, Schematic organization of the newly discovered alternatively spliced ZmPSY1 transcript, T003. Probe used in C is complementary to the common region to the ZmPSY1 transcripts. C, RNA gel blot analysis of ZmPSY1 transcripts responding to SCMV infection at 5 and 9 dpi. This experiment was repeated twice with similar results. D and E, Relative expression levels of ZmPSY1 total transcripts (D) and T003 (E) were separately determined using RT-qPCR of RNA in the first systemically infected leaf from SCMV-infected and the equivalent leaf from mock-inoculated maize seedlings at 3, 5, 7, 9, and 12 dpi. Data were presented as means ± se (n = 6). Asterisks indicate significant differences between values for mock-inoculated and SCMV-infected plants at the same time point as evaluated by two-tailed Student’s t tests. F and G, The protein levels of ZmPSY1 were analyzed by immunoblotting at 3, 5, 7, 9, and 12 dpi in SCMV-infected plants or equivalent samples from mock-inoculated plants. The arrowhead indicates the position of the ZmPSY1 band in (F). The relative amount of ZmPSY1 (G) was calculated using the software ImageJ, and the lanes of 3-dpi mock were set to 1.0. Actin was used as gel loading control. Data were presented as means ± se (n = 3). P values evaluated by two-tailed Student’s t test analysis indicated no significant differences between the relative ZmPSY1 levels for mock-inoculated and SCMV-infected plants within time points.

Figure 4.

Mutation of the ZmPSY1 gene ameliorates SCMV-induced pathogenesis. A, Schematic representation of the ZmPSY1 transcript T001, annotated with the positions of mutations in the two psy1 mutant maize lines d13 and d14. Red rectangles and interconnecting black broken lines represent coding region and introns, respectively, and red outlined boxes indicate UTRs. B and C, the left shows mosaic/streaking symptoms in systemically infected leaves of SCMV-infected seedlings of the d13 and d14 mutant lines and those in wild-type (WT) seedlings at 5 dpi (B) and 9 dpi (C). Middle representation in (B) and (C), accumulation of viral RNA was measured in wild-type, d13, and d14 leaves by RT-qPCR using ZmUbi as the internals standard. Error bars represent means ± se (n = 3). Asterisks indicated significant difference as evaluated by two-tailed Student’s t test analysis. The right shows immunoblot analysis of accumulation levels of SCMV CP in the first systemically infected leaf of wild-type, d13, and d14 seedlings. Arrowheads indicate the position of SCMV CP. The relative intensity of each band detected with anti-CP was quantified and the level of CP in wild-type plants was taken to be 1.0. Values (means ± se) are graphically presented on a histogram (n = 3). The different letters above each bar in the right of B and C indicate statistically significant differences as determined by a one-way ANOVA followed by Tukey’s multiple test (P < 0.05). Scale bars = 0.5 cm. D, TEM of chloroplasts in the first systemically infected leaves of mock-inoculated (left) and SCMV-infected (center) wild-type, d13, and d14 seedlings at 9 dpi. Grana are indicated with red arrows and their thicknesses were quantified for chloroplasts in both mock-inoculated and SCMV-infected plants (histograms to the right of the electron micrographs). Formation of peripheral vesicles (red arrowheads) was only observed in chloroplasts of SCMV-infected wild-type plants but not in SCMV-infected d13 and d14. Data are presented as means ± se (n = 9 or 10 chloroplasts). Significant differences were identified by a one-way ANOVA followed by Tukey’s multiple test and are shown by the different letters above each bar (P < 0.05). Scale bars = 0.5 μm.

Figure 5.

The ZmPSY1 T001 3′ UTR enhances translation efficiency more than that of T003. A, RNA structural analyses of the 3′UTRs of ZmPSY1 T001 (1,349 to 1,776 nucleotides) and T003 (1,349 to 2,121 nucleotides). The structures were colored by base-pairing probabilities (from 0 to 1). For unpaired regions, the colors denoted the probability of being unpaired. The 3′-UTRs are shown in blue boxes. B, Sensor constructs comprising the EGFP-coding sequence fused to 3′-UTRs. The EGFP coding sequence was fused with the native 3′-UTRs (construct T001-3′-UTR and T003-3′-UTR). 35S, CaMV 35S promoter; NOS, NOS terminator. C to G, EGFP constructs were transiently expressed in N. benthamiana leaves and examined via observation using a confocal microscope with the same laser parameters for EGFP fluorescence (C and D), by immunoblot analysis (E and F), and by RT-qPCR analysis (G) of leaves infiltrated with the sensor constructs. EGFP fluorescence was quantified in (D) using the LAS Application Suite X imaging system (Leica; n = 14 leaves from three biological replicates). Asterisks in (D) and (F) indicate significant differences evaluated by two-tailed Student’s t tests. Error bars are means ± se. Quantification of EGFP mRNA levels was performed with five independent experiments (n = 5); error bars are means ± se (G). The relative intensities of bands detected by immunoblotting were quantified using the software ImageJ, and the value for T001 in each biological repeat (n = 3) was arbitrarily set to 1.0. Gels were stained with Coomassie Blue R-250 as loading control; band of the large subunit of Rubisco is shown. Scale bar = 1.0 cm on the leaf image; scale bars = 250 μm in microscopic images. H and I, Reporter transcripts bearing ZmPSY1 T001-3′-UTR and T003-3′-UTR exert different translation efficiency in the wheat germ in vitro translation system. The EGFP (including stop codon) with variant 3′-UTRs of ZmPSY1 transcripts were separately transcribed in vitro. In vitro translation was performed in a wheat germ lysate system with equimolar amounts (0.01 nmol) of mRNA species at 25°C for 1 h. After boiling in SDS sample buffer, translation products were analyzed by immunoblotting. (−) Negative control, adding sterile water in wheat germ lysate system; (+) positive control at a 1:1,000 dilution, N. benthamiana leaves transiently over-expressing EGFP with no UTR construct. Red arrowhead indicates the bands of EGFP. Gels were stained with Coomassie blue R-250 as loading control. The relative intensity of each band detected with anti-EGFP was quantified and the level of the EGFP band for T001-3′-UTR taken to be 1.0. Values were presented as means ± se (n = 3). Significant differences were identified using two-tailed Student’s t tests.

Figure 6.

VIGS-mediated knockdown of ZmPSY1 transcripts significantly decreases SCMV accumulation. A, Location of sequences subcloned into the CMV vector to stimulate VIGS of all ZmPSY1 transcripts (CMV-ZmPSY1) or only of the alternatively spliced transcript T003 (CMV-T003). Red rectangles and interconnecting black broken lines represent coding region and introns, respectively; black blank boxes show UTRs; green and blue boxes sequences are complementary to target sequences. Arrows marked QF4/QR4 and QF10/QR10 show the positions of RT-qPCR primers used in C and D. B to G, Silencing of ZmPSY1 and T003 decreased SCMV infection, viral RNA, and CP accumulation. RT-qPCR assays were used to quantify ZmPSY1 transcripts and SCMV RNA accumulation in the first systemically infected leaves of silenced seedlings and control seedlings infected with CMV-GFP (C–E). Data in C to E are presented as means ± se (n = 5). Significant differences were identified by one-way ANOVA followed by Tukey’s multiple test. The different letters above each bar indicate statistically significant differences (P < 0.05). Immunoblot analysis of CP (band indicated with a black arrowhead) accumulation in F and G. The relative intensity of each CP band was quantified. Error bars represent means ± se (n = 3). Statistical differences were determined by two-tailed Student’s t tests. Scale bar = 1.0 cm.