Transcriptomic changes associated with infection of Nicotiana benthamiana plants with tomato ringspot virus (genus Nepovirus) during the acute symptomatic stage and after symptom recovery

Abstract

We have characterized the transcriptome of Nicotiana benthamiana plants infected with tomato ringspot virus (ToRSV), a nepovirus. We analyzed two different stages of infection: the acute systemic symptomatic stage and the symptom recovery stage in which young leaves emerge without visible symptoms. In agreement with previous observations, we note a similar concentration of viral RNAs in symptomatic and recovered leaves. Extensive reprogramming of the plant transcriptome was observed in symptomatic leaves, including upregulation of genes characteristic of biotic stress responses and downregulation of genes associated with the function and translation of chloroplasts. The majority of plant genes that were differentially regulated during the symptomatic stage returned to their basal levels after recovery. Thus, the extent of changes in the plant transcriptome was correlated with symptom intensity. However, we also identified genes that remained upregulated after the symptomatic stage or that were specifically induced at the symptom recovery stage. The list of genes that were upregulated at the symptom recovery stage was diverse and included several types of cysteine-rich antimicrobial peptides, notably two defensin-like genes that were specifically upregulated in recovered leaves, as confirmed by droplet-digital PCR. Several plant miRNAs were also differentially expressed in ToRSV-infected plants. Notably, miR391 was upregulated at both stages of infection, whereas miR530 and miR1919 were specifically upregulated during the symptomatic stage of infection. Several predicted miRNA targets were differentially regulated in our dataset, including new targets as well as previously validated targets (e.g., zinc finger A20/AN1 domain-containing stress-associated protein 1, a known target of miR530). Many of the miRNA predicted targets were related to plant defense responses and may contribute to symptom induction and/or symptom recovery.

Fig 1. Typical ToRSV-induced symptoms and sampling method.

(A) Schematic diagram illustrating the sampling method. At 3 dpi, ToRSV-inoculated N. benthamiana plants exhibit localized necrotic rings on the lower inoculated leaves (red arrow), and vein-clearing symptoms on the upper systemically infected leaves (orange arrow). Samples were taken from upper systemically infected symptomatic leaves (orange arrow) or equivalent leaves (light-yellow arrow) from mock-inoculated plants at 3 dpi. (B) Representative pictures of a ToRSV-infected plant and a mock-inoculated plant at 3 dpi. Leaves are marked as in (A), with typical sampled leaves shown by the orange and light-yellow arrows. (C) Representative picture of a ToRSV-infected plant at the recovery stage alongside a mock-inoculated plant (picture taken at 11 dpi). Typical sampled leaves are marked with the blue arrows. Although ToRSV-infected plants are stunted compared to mock-inoculated plants, the upper recovered leaves are asymptomatic.

Fig 2. Principal component analysis plot of (A) total RNA and (B) small RNA sequencing data.

Sequences from repeats 1, 2 and 3 are color coded in black, blue and red, respectively. The numbers beside each data point indicates the number of days post-inoculation (3 dpi or 10 dpi) for mock-inoculated samples (+) or ToRSV-inoculated samples (•).

Fig 3. Mapping of virus-derived small RNAs to the viral genome.

The coding regions for protein domains in the RNA1 and RNA2 polyproteins are shown with the boxes. RNA1 and RNA2 share 81% nucleotide sequence identity on their 3’ UTRs (red bar). The 5’ UTRs and the 5’ ends of the X1 and X3 coding regions of RNA1 and RNA2, respectively, also share extensive nucleotide sequence identity (dark red bar). NTB: putative nucleoside triphosphate binding protein, VPg: virus genome-linked protein, Pro: protease, Pol: RNA-dependent RNA polymerase, MP: movement protein, CP: coat protein. The functions of proteins X1, X2, X3 and X4 are not known.

Fig 4. Differential expression of plant genes and miRNAs during ToRSV-Rasp1 infection.

(A) Venn Diagram showing differentially expressed genes (1,263 DEGs). (B) Venn diagram showing differentially expressed miRNA (9 miRNAs). Only DEGs or miRNAs with absolute fold changes ≤ 0.5 or ≥ 2, p-value ≤ 0.05 and maximum group mean ≥ 5 are included in the analyses. (C) Volcano plot displaying DEGs in four pairwise comparisons. RNA sequencing reads mapped to N. benthamiana were analyzed and pairwise comparisons were made as indicated below each panel. DEGs with max group mean value < 5 were excluded from the analysis. The red bar is set at –log10 (p-value) of 1.3 (corresponding to a p-value of 0.05). Dots above the bar are differentially expressed in a significant manner. All dots above the bars also corresponded to DEGs with absolute fold changes ≤ 0.5 or ≥ 2.

Fig 5. SEA analysis for DEGs that are induced or repressed in the symptomatic vs mock (Sympt vs Mock) or recovered vs symptomatic (Rec vs Sympt) comparisons.

Circle size and colors indicate level of enrichment and FDR adjusted P values, respectively.

Fig 6. Quantification of ToRSV RNA1 (blue) and RNA2 (orange) in total RNA samples extracted from symptomatic leaves at 3 dpi (S 3) or recovered leaves at 10 dpi (R 10).

(A) Total RNA samples were diluted 1:2,000 and 3 ul of the solution was tested by ddPCR. Values obtained were normalized using the two reference genes PP2A and actin. (B) Ratio of RNA2 to RNA1 was calculated using the values in (A).

Fig 7. Quantification of selected plant mRNAs using ddPCR.

Total RNA samples extracted from mock-inoculated leaves at 3 dpi (M 3) or 10 dpi (M 10) or from symptomatic (S 3) or recovered leaves (R 10) were quantified by ddPCR. Total RNAs were diluted 1:20 prior to the ddPCR reaction. Exceptions were DFL (1:5 dilution), HSP70 and PPctI (1:100 dilution for RNAs for S3 samples), and SAR8 (1:200 dilution for RNAs for S3 and R10 samples). Values were normalized using the two reference genes PP2A and actin. Values are reported as fold change compared to the M3 sample (set at 1) for each biological repeat. HSP70: heat shock protein 70; PPctI-D: Peptidyl-prolyl cis-trans isomerase D (cyclophilin); SAR8: mRNA inducible by salicylic acid or by TMV during systemic acquired resistance; MORN3: MORN-motif repeat protein similar to AT1G21920.1 MORN3 (MRF1) protein; AP-4: Transcription factor AP-4; SP1: signal peptidase 1, TIM: Mitochondrial import inner membrane translocase subunit Tim17/Tim22/Tim23 family; FREE: Zinc finger FYVE domain-containing protein 21; IAA4: Auxin-responsive protein IAA4; AMD: Amidotransferase; DFL: defensin-like protein.