Synergistic infection of two viruses MCMV and SCMV increases the accumulations of both MCMV and MCMV-derived siRNAs in maize

Abstract

The co-infection of Maize chlorotic mottle virus (MCMV) and Sugarcane mosaic virus (SCMV) can cause maize lethal necrosis. However, the mechanism underlying the synergistic interaction between these two viruses remains elusive. In this study, we found that the co-infection of MCMV and SCMV increased the accumulation of MCMV. Moreover, the profiles of virus-derived siRNAs (vsiRNAs) from MCMV and SCMV in single- and co-infected maize plants were obtained by high-throughput sequencing. Our data showed that synergistic infection of MCMV and SCMV increased remarkably the accumulation of vsiRNAs from MCMV, which were mainly 22 and 21 nucleotides in length. The single-nucleotide resolution maps of vsiRNAs revealed that vsiRNAs were almost continuously but heterogeneously distributed throughout MCMV and SCMV genomic RNAs, respectively. Moreover, we predicted and annotated dozens of host transcript genes targeted by vsiRNAs. Our results also showed that maize DCLs and several AGOs RNAs were differentially accumulated in maize plants with different treatments (mock, single or double inoculations), which were associated with the accumulation of vsiRNAs. Our findings suggested possible roles of vsiRNAs in the synergistic interaction of MCMV and SCMV in maize plants.

Figure 1. Co-infection of SCMV and MCMV increased the accumulation of MCMV.

(A,B) The symptoms of the first systemically infected leaves at 9 and 10 dpi, respectively. (C) The accumulations of MCMV genome were determined by Northern blotting at 9 dpi in buffer (Mock), SCMV, MCMV and S + M inoculated maize plants. Three independent MCMV and S + M infected maize plants were used, and Mock and SCMV inoculated plants were used as controls. Methylene blue staining (bottom panel) of the same extracts was shown to demonstrate equal loading. (D) The relative expressions of SCMV RNAs were determined by qRT-PCR at 9 dpi in SCMV and S + M infected maize plants. Three independent experiments were conducted with at least 3 biological replicates each and the data were analysed using a two-sample t-test. Bars represented the grand means ± SD. Different letters in lowercase indicate a significant difference (P-value < 0.05). (E,F) The accumulation levels of MCMV and SCMV CP, respectively. Western blotting was performed using the systemically infected leaves of buffer (Mock), SCMV, MCMV or S + M inoculated maize plants at 9 dpi. Coomassie brilliant blue (CBB) staining (bottom panel) of the same extracts was shown to demonstrate equal loading.

Figure 2. Size distribution of vsiRNAs.

(A) Size distribution of M-vsiRNAs in MCMV and S + M inoculated maize plants. (B) Size distribution of S-vsiRNAs in SCMV and S + M inoculated maize plants.

Figure 3. Percentage distribution of vsiRNAs with respect to strand polarity.

(A) The strand polarity of M-vsiRNAs in MCMV and S + M inoculated maize plants. (B) The strand polarity of S-vsiRNAs in SCMV and S + M inoculated maize plants.

Figure 4. 5′-terminal nucleotide abundance of vsiRNAs.

(A) 5′-terminal nucleotide abundance of M-vsiRNAs in MCMV and S + M inoculated maize plants. (B) 5′-terminal nucleotide abundance of S-vsiRNAs in SCMV and S + M inoculated maize plants.

Figure 5. The single-nucleotide resolution maps of 21- and 22-nt vsiRNAs.

(A) Schematic diagram of the MCMV genome. (B) Schematic diagram of the SCMV genome. The single-nucleotide resolution maps of 21- and 22-nt M-vsiRNAs along the MCMV genome in MCMV (C) and S + M (E) inoculated maize plants and 21- and 22-nt S-vsiRNAs along the SCMV genome in SCMV (D) and S + M (F) inoculated maize plants. The bars above the axis represent the reads of vsiRNAs from the viral genomic strand starting at the respective positions; the bars below represent the reads of vsiRNAs from the complementary strand of viral genomes ending at the respective positions.

Figure 6. GO classification of the predicted target genes of vsiRNA derived from MCMV and SCMV in maize.

The vsiRNA target genes were assigned using Blast2GO. “Control” indicates the percentage of the genes with specific category in all GO-annotated maize genes.

Figure 7. KEGG classification of the predicted target genes of vsiRNAs derived from MCMV and SCMV in maize.

The vsiRNA target genes were assigned based on the KEGG database using BLASTx. I: Metabolism; II: Genetic Information Processing; III: Environmental Information Processing; IV: Cellular Processes; V: Organismal Systems; VI: Human Diseases. “Control” means the percentage of the genes with specific category in all KEGG-annotated maize genes.

Figure 8. Northern blotting analysis of vsiRNAs.

(A,B) Northern blotting analysis of M-vsiRNAs in MCMV and S + M inoculated maize plants. (C) Northern blotting analysis of S-vsiRNAs in SCMV and S + M inoculated maize plants. The “M” means the vsiRNAs from MCMV and “S” means that from SCMV. The numbers represent vsiRNAs starting positions of the (+)-sense strand or ending positions of the (−)-sense strand of viral genomes. “(+)” indicates vsiRNAs derived from (+)-sense strand of viral genomes and “(−)” indicates the (−)-sense strand. “21” and “22” means the positions of 21-nt and 22-nt vsiRNAs, respectively. U6 was used as a loading control.

Figure 9. The expression levels of maize DCLs and several AGOs mRNAs in buffer (Mock), SCMV, MCMV and S + M inoculated maize plants.

The expression levels were determined by qRT-PCR at 9 dpi. Three independent experiments were conducted with at least 3 biological replicates each and the data were analysed using a two-sample t-test. Bars represented the grand means ± SD. Lowercase letters indicate significant difference (P-value < 0.05).