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. 2022 Mar 3;14(3):523.
doi: 10.3390/v14030523.

Transcriptome and Small RNA Profiling of Potato Virus Y Infected Potato Cultivars, Including Systemically Infected Russet Burbank

Brian T Ross  1 , Nina Zidack  2 , Rose McDonald  1 , Michelle L Flenniken  1
Affiliations

Transcriptome and Small RNA Profiling of Potato Virus Y Infected Potato Cultivars, Including Systemically Infected Russet Burbank

Brian T Ross et al. Viruses. .

Abstract

Potatoes are the world's most produced non-grain crops and an important food source for billions of people. Potatoes are susceptible to numerous pathogens that reduce yield, including Potato virus Y (PVY). Genetic resistance to PVY is a sustainable way to limit yield and quality losses due to PVY infection. Potato cultivars vary in their susceptibility to PVY and include susceptible varieties such as Russet Burbank, and resistant varieties such as Payette Russet. Although the loci and genes associated with PVY-resistance have been identified, the genes and mechanisms involved in limiting PVY during the development of systemic infections have yet to be fully elucidated. To increase our understanding of PVY infection, potato antiviral responses, and resistance, we utilized RNA sequencing to characterize the transcriptomes of two potato cultivars. Since transcriptional responses associated with the extreme resistance response occur soon after PVY contact, we analyzed the transcriptome and small RNA profile of both the PVY-resistant Payette Russet cultivar and PVY-susceptible Russet Burbank cultivar 24 hours post-inoculation. While hundreds of genes, including terpene synthase and protein kinase encoding genes, exhibited increased expression, the majority, including numerous genes involved in plant pathogen interactions, were downregulated. To gain greater understanding of the transcriptional changes that occur during the development of systemic PVY-infection, we analyzed Russet Burbank leaf samples one week and four weeks post-inoculation and identified similarities and differences, including higher expression of genes involved in chloroplast function, photosynthesis, and secondary metabolite production, and lower expression of defense response genes at those time points. Small RNA sequencing identified different populations of 21- and 24-nucleotide RNAs and revealed that the miRNA profiles in PVY-infected Russet Burbank plants were similar to those observed in other PVY-tolerant cultivars and that during systemic infection ~32% of the NLR-type disease resistance genes were targeted by 21-nt small RNAs. Analysis of alternative splicing in PVY-infected potato plants identified splice variants of several hundred genes, including isoforms that were more dominant in PVY-infected plants. The description of the PVYN-Wi-associated transcriptome and small RNA profiles of two potato cultivars described herein adds to the body of knowledge regarding differential outcomes of infection for specific PVY strain and host cultivar pairs, which will help further understanding of the mechanisms governing genetic resistance and/or virus-limiting responses in potato plants.

Keywords: Potato virus Y; alternative splicing; extreme resistance; small RNAs; tolerance.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Transcriptome level evaluation of the response of two potato cultivars indicates that response to Potato virus Y-inoculation varies by cultivar and time point. (A) Schematic representation of the experiments performed to investigate potato antiviral immune responses. Potato plants were infected with PVY or mock-infected (n = 6 plants per treatment, per time point, per biological replicate). Leaf tissue samples were collected from the inoculation point at 24 hours post-infection (hpi) for both cultivars and at 1- and 4-weeks post-infection (wpi) in PVY-susceptible Russet Burbank plants. RNA was extracted from leaf tissue samples and prepared for either qPCR or high throughput sequence analysis. qPCR was used to quantify PVY abundance in all plants and to validate differential gene expression results for a subset of genes in a subset of plants from all three biological replicates. (B) Potato transcriptional responses to PVY-infection are time- and cultivar-dependent. The Venn diagram illustrates the number of unique and shared differentially expressed genes (DEGs) in Payette Russet (blue) and Russet Burbank (shades of green) plants in response to PVY infection. No differentially expressed genes were shared between all time points and cultivars, though hundreds of differentially expressed genes were shared between Payette Russet at 24 hpi and Russet Burbank at 1 and 4 wpi. Full lists of differentially expressed genes and their corresponding fold changes are included in Supplemental File S1.
Figure 2
Figure 2
Select genes that exhibited notable changes in expression in PVY-infected potato plants relative to mock-inoculated plants. Table highlights a small subset of DEGs identified by transcriptomic analysis; the relative expression of these genes of interest varies by time point (24 hpi, 1 and 4 wpi) and potato cultivar (Payette Russet and Russet Burbank). Highlighting indicates lower (yellow) or greater (blue) expression in virus-infected plants as compared to mock-infected control plants and “N” is used for non-detectable and/or non-statistically significant values.
Figure 3
Figure 3
Potato transcriptome data supported by quantitative PCR analysis. Quantitative PCR (qPCR) was used to validate the differential expression analysis results for a subset of genes for each time point (24 hours post-inoculation, 1- and 4- weeks post-inoculation) and cultivar (PVY-resistant Payette Russet, PVY-susceptible Russet Burbank) used in this study. (A) qPCR assessment of the relative expression of Dicer2-like (Soltu.DM.11G004150), TCP (Soltu.DM.04G011720), and NPR1-like (Soltu.DM.07G014680) in PVY-inoculated Payette Russet leaves at 24 hpi relative level in mock-infected control plants, determined that their expression was similar (0.04 log2 fold change), higher (1.10 log2 fold change), and lower (−1.11 log2 fold change), respectively. (B) Evaluation of the expression of Dicer2-like, MLP, and NPR1-like in PVY-infected Russet Burbank plants at 24 hpi relative to uninfected controls indicated that only the expression of MLP (Soltu.DM.04G002870) was reduced by PVY-infection (−0.84 log2 fold change). (C) The qPCR gene expression results from upper, uninoculated leaves of PVY-infected Russet Burbank plants at 1 wpi compared to mock-infected controls indicate a reduced expression of Dicer4-like (Soltu.DM.07G000040) (−1.35 log2 fold change) and NPR1-like (−0.98 log2 fold change), and a non-statistically significant increase in the expression of a putative mitochondrial RNA helicase expression (Soltu.DM.12G023910) (0.32 log2 fold change). (D) The qPCR gene expression results from upper, uninoculated leaf samples of PVY-infected Russet Burbank plants at 4 wpi compared to uninfected control plants indicated that the expression of Dicer2-like (1.22 log2 fold change) and AGO1-like (Soltu.DM.03G019130) (0.92 log2 fold change) was increased, whereas the NPR1-like expression (−0.83 log2 fold change) was reduced. Statistical differences in gene expression between mock-infected and PVY-infected potato plants (n = 6) were performed using Wilcoxon rank sum test, * p < 0.05. All of the qPCR results mirrored the transcriptome level assessment results, except that the increased expression of RNA helicase in PVY-infected Russet Burbank plants was not statistically significant by qPCR whereas it was in the transcriptome data. This figure includes data from one representative biological replicate and the results from all three biological replicates are included (Supplemental Figures S1 and S2).
Figure 4
Figure 4
Potato small RNA transcriptional response to PVY-infection is time- and length-dependent. A Venn diagram depicts unique and shared differentially expressed 21- and 24-nucleotide small RNAs in Russet Burbank at 1 wpi (blue) and at 4 wpi (yellow) in response to PVY infection. No differentially expressed small RNAs were shared between all of the time points and cultivars. Over half of the 21-nucleotide small RNAs that increased in abundance at 1 wpi were also differentially expressed at 4 wpi. Most of the differentially expressed small RNAs at 1 wpi were 24 nucleotides long, while most of those differentially expressed at 4 wpi were 21 nucleotides long, with many fewer 24-nucelotide small RNAs at that time point. Gene ontology results indicate that defense response and ADP-binding were enriched among all differentially expressed 21-nucleotide small RNAs, while enrichment for photosynthesis, ribosome- and translation-associated genes were enriched in the 21-nucleotide small RNAs at 4 wpi only. Transporter activity, carbohydrate metabolism, and the cell wall were enriched among 24-nucleotide small RNAs at 1 wpi, with the photosynthetic membrane, ADP-binding, and the ribosomes were enriched among 24-nucleotide small RNAs at 4 wpi. Full lists of differentially expressed genes and their corresponding fold changes are reported in Supplemental File S2.
Figure 5
Figure 5
Phased short-interfering RNAs predominantly target NLR proteins in Russet Burbank and are not correlated with expression patterns of mRNAs. (A) PhasiRNAs separated by time point and gene type. Approximately half (25/52 at 1 wpi, 48/93 at 4wpi) of the phasiRNAs aligned to annotated NLR resistance genes at each time point (blue); whereas clusters that did not align to NLR resistance genes are in yellow. (B) Similar phasiRNAs are shared between time points and among small RNA lengths. All but one of the 21-nucleotide phasiRNAs identified at 1 wpi was also identified at 4 wpi (blue). Similarly, all seven 24-nucleotide phasiRNAs at 4 wpi were also identified as phasiRNAs at 1 wpi (yellow). (C,D) Normalized expression of genes targeted by phasiRNAs colored by state of differential expression. Genes targeted by phasiRNAs do not exhibit a correlation with either increased or decreased expression. Full lists of differentially expressed phasiRNAs and their corresponding fold changes are reported in Supplemental File S2.
Figure 6
Figure 6
Alternative splicing analysis in Payette Russet and Russet Burbank potato cultivars in response to PVY infection. Hundreds of genes differentially spliced genes were identified in this study, some of which shared splicing patterns between time points. Gene ontology analysis of the spliced genes revealed glucose-6-phosphate enrichment among differentially spliced genes in Payette Russet at 24 hpi. The highest number of differential splicing occurred at 1 wpi in Russet Burbank, with over 200 genes that were likely differentially spliced and ontology enrichment for mRNA binding/processing, chloroplast, and the citrate cycle.
Figure 7
Figure 7
Alternative splicing profiles of select genes in PVY-infected Russet Burbank plants. (A) Isoform 2 of the gene Polynucleotidyl transferase, ribonuclease H-like superfamily protein (Soltu.DM.04G031630) exhibited increased usage in PVY-infected Russet Burbank plants at 1 wpi, resulting in a higher proportion of transcripts encoding a truncated C-terminal end, which may be important for an effective RNA interference response. (B) Isoform 2 of the gene Syntaxin of plants (Soltu.DM.10G025490) exhibits increased usage relative to isoform 1 in PVY-infected Russet Burbank plants at 4 wpi. (C) Isoform 2 of the gene Basic-leucine (bZIP) transcription factor family protein (Soltu.DM.02G015010), which contains an intrinsically disordered domain, exhibits increased usage at 4 wpi in PVY-infected Russet Burbank plants.
Figure 8
Figure 8
Poly(A) polymerase exhibits alternative splicing at 24 hpi, 1 wpi, and 4 wpi in PVY-infected Russet Burbank plants. Transcript isoforms of the potato gene, poly(A) polymerase (Soltu.DM.01G018800) exhibit similar differential splicing patterns in all sampled time points of Russet Burbank during PVY infection. (A) A splicing graph depicting the four different isoforms of poly(A) polymerase expressed in Russet Burbank. Isoform 2 contains an NTP transferase 2 domain and isoform 4 contains a signal peptide domain. (B) Gene expression, isoform expression, and isoform usage graphs for poly(A) polymerase at 24 hpi, 1 wpi, and 4 wpi in Russet Burbank plants. All four isoforms were expressed at 24 hpi, 1 wpi, and 4 wpi. Isoform 2 contains an NTP transferase 2 domain. Poly(A) polymerase is differentially expressed at 4 wpi but not at 24 hpi or 1 wpi. This increase in expression is likely due to an increase in expression of isoform 3, which also exhibits a significant increase in isoform usage at 4 wpi. In this figure * p < 0.05; *** p < 0.0005; and “ns” indicates non-significant.
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