HTS analysis of resistance induction against PPV by four hairpin constructs in Nicotiana benthamiana Domin

Abstract

Plum pox virus (PPV) is the most devastating viral disease of the stone fruits worldwide. Inefficiency of the traditional control measures against PPV along with its globally widespread distribution and the economic importance of stone fruits, signify the necessity and importance of PPV resistance programs. In the present study, Agrobacterium-mediated transformation of Nicotiana benthamiana Domin was performed using four inverted repeat constructs derived from UTR/P1, HCPro, HCPro/P3, and CP regions of PPV-T isolate KyEsAp301. The efficacy of the constructs for inducing virus resistance in transgenic plants was evaluated by inoculation with PPV-D, -M, and -T strains. The potential of hairpin structures in the production of siRNAs and miRNAs in both wild-type and transgenic plants was compared by small RNA high-throughput sequencing. Although the four PPV genomic regions were used for transgenic resistance in previous experiments, small RNA high-throughput sequencing was first time used in this study to demonstrate the efficacy of the PPV constructs and to determine expression profiles of siRNAs and miRNAs. The results revealed that the potentials of hairpin constructs in producing siRNAs and their accumulation in target regions were significantly different. Expression profiles of several known and novel miRNAs were dramatically changed in response to PPV infection in both wild-type and transgenic plants, demonstrating plausible involvement of these miRNAs in plant-virus interactions. Based on the abundance of siRNAs and lack of PPV virus accumulation in transgenic plants harboring UTR/P1 and CP hairpin construct, we have concluded that UTR/P1 and CP are likely the best viral regions for induction of resistance against PPV.

Keywords: Potyvirus; RNAi; Small RNA sequencing; Virus silencing.

Fig. 1

Different stages of regeneration of transgenic plants. (a-c) Generation of transgenic shoots from explants. (d) Transgenic plants growing in turf. (e) inoculum source for PPV-D, -M, and -T strains. (f) Amplification of genes of interest for hairpin construction. Lanes 1: UTR/P1 752pb, 2: HC-Pro 649 bp, 3: HC-Pro/P3 594 bp and 4: CP 990 bp. M: 100 bp DNA ladder. (g) Agarose gel electrophoresis of RT-PCR products of 5 selected plants harboring four hairpin constructs showing resistance. Lanes 1 to 5: Plants with UTR/P1 hairpin construct; Lanes 6 to 10: Plants with HC-Pro/P3 hairpin construct; Lanes 11 to 15: Plants with HCPro hairpin construct; Lanes 16 to 20: Plants with CP hairpin construct. M: 100 bp DNA ladder. The images (f and g) depict agarose gel electrophoresis results from RT-PCR experiments. The presented bands accurately represent the PCR products without any manipulation. While minor adjustments may have been made to align the panels within the figure layout, no significant cropping affecting the integrity of the data was performed. The images retain the original banding patterns, ensuring that the necessary regions are visible in a single, consistent line for proper interpretation.

Fig. 2

Length distribution of counts of (a) total, (b) unique small non-coding RNAs (sRNA), (c) miRNAs, and (d) siRNAs.

Fig. 3

Size distribution and Circos plot of siRNA. (a) Size distribution of the 21–25 nucleotide siRNAs in transgenic plant harboring UTR/P1, HC-Pro, HC-Pro/P3 and CP hairpin constructs, and (b) Circos plot of the normalized (reads per million) from outside to inside circles: UTR/P1 (green), HC-Pro (orange), HC-Pro/P3(pink), CP (blue), Infected -wild type(brown), uninfected (teal).

Fig. 4

Venn diagram showing differentially expressed miRNA comparisons. (a) Comparison of miRNAs between each transgenic line harboring UTR/P1, HC-Pro, HC-Pro/P3, CP hairpin constructs, and PPV positive (WT+) and PPV negative (WT-) samples. (b) Comparison of miRNAs among transgenic lines harboring UTR/P1, HC-Pro, HC-Pro/P3, CP hairpin constructs.

Fig. 5

Gene ontology (GO) enrichment analysis of miRNAs’ target genes, showing the most significantly enriched GO terms of the miRNA target genes in the cellular components, molecular function, and biological processes.