Enhancing Pseudomonas syringae pv. Actinidiae sensitivity in kiwifruit by repressing the NBS-LRR genes through miRNA-215-3p and miRNA-29-3p identification

Abstract

Kiwifruit bacterial canker, caused by Pseudomonas syringae pv. actinidiae (PSA), poses a grave threat to the global kiwifruit industry. In this study, we examined the role of microRNAs (miRNAs) in kiwifruit's response to PSA. Kiwifruit seedlings subjected to PSA treatment showed significant changes in both miRNA and gene expression compared to the control group. We identified 364 differentially expressed miRNAs (DEMs) and 7170 differentially expressed genes (DEGs). Further analysis revealed 180 miRNAs negatively regulating 641 mRNAs. Notably, two miRNAs from the miRNA482 family, miRNA-215-3p and miRNA-29-3p, were found to increase kiwifruit's sensitivity to PSA when overexpressed. These miRNAs were linked to the regulation of NBS-LRR target genes, shedding light on their role in kiwifruit's defence against PSA. This study offers insights into the miRNA482-NBS-LRR network as a crucial component in enhancing kiwifruit bioresistance to PSA infestation and provides promising candidate genes for further research.

Keywords: DEMs and DEGs; Kiwifruit bacterial canker; NBS-LRR; PSA; miRNA-215-3p and miRNA-29-3p.

Figure 1

Screening of PSA-responsive miRNAs. Expression levels of 25 miRNAs were examined by qRT-PCR under PSA treatment for 12 h and 48 h. The expression level of each miRNA was calculated relative to that of the respective untreated control samples. Kiwi Ac-AcUin (FG520231) was used as an internal control to normalise the expression data. Different colours represent different treatments. X-axis red markers represent screened candidate PSA-responsive miRNAs and error bars represent standard deviations calculated based on three biological replicates.

Figure 2

Validation of 5 miRNAs for PSA response functions by transient overexpression analysis in kiwifruit leaves. (A) Schematic diagram of the gene cassette used for the overexpression of 5 miRNAs in kiwifruit. (B) The relative expression level of the 5 miRNA was calculated relative to the expression in the respective untreated control samples (0 h). Kiwifruit Ac-AcUin (FG520231) was used as an internal control to normalize the expression data. Different colours represent different treatments. The error bars represent the standard deviation calculated based on three biological replicates. (C–H) After transient overexpression of the 5 miRNAs in kiwifruit leaves, photographs of plant leaves after placenta blue staining and decolourisation were performed on the 8th day after PSA treatment. Scale bars = 1 cm. (I) Disease index (%) at 8 days after PSA inoculation. Control: transient overexpression of empty vector. (J) Relative lesion diameter. Data are mean ± SE of three independent experiments. Double asterisks indicate highly significant differences between samples.

Figure 3

Functional validation of miRNA-29-3p and miRNA-215-3p transgenic kiwifruit against PSA infection. (A, B) Quantitative real-time PCR analysis of the abundance of miRNA-29-3p and miRNA-215-3p in WT, miRNA-29-3p overexpressing (A), and miRNA-215-3p overexpressing (B) kiwifruit lines. (C–J) The stem phloem of 1-year-old transgenic plants was treated with PSA, and after 14 days, the phloem tissue was scraped off to take photographs (scale bar = 1 cm). (K, L) Infection site assays on kiwifruit plants 14 days after infection with PSA showed that WT and overexpressing kiwifruit lines had disease indices and relative lesion diameter. (M) Transcript accumulation of PSA avrE1 gene in these inoculated plants 24 h after inoculation with PSA. (N) MDA content of the leaves from WT, and overexpressing lines at 14 days after inoculation with PSA. Data are the means ± SEs from three independent experiments. Double asterisks indicate highly significant differences between samples.

Figure 4

Screening of NBS-LRR target genes (named disease resistance protein, DRP) regulated by miRNA-215-3p and miRNA-29-3p. (A) Prediction of DRPs regulated by miRNA-215-3p and miRNA-29-3p, and they regulate 29 and 7 potential DRPs (blue), respectively, and co-regulate 5 DRPs (red). (B–F) Expression of 5 DRPs was analysed by qRT-PCR in overexpressing miRNA-215-3p and miRNA-29-3p strains 24 h after inoculation with PSA. The relative expression levels of each DRP were calculated relative to the expression levels of the WT samples. Kiwi Ac-AcUin (FG520231) was used as an internal control to normalise the expression data. Error bars represent standard deviations calculated based on three biological replicates. Double asterisks indicate highly significant differences between samples.

Figure 5

Expression pattern analyse of these four DRPs under PSA treatment. (A–D) The expression levels of the four DRPs in kiwifruit leaves were measured by qRT-PCR from 1 to 5 dpi after PSA spraying. Data are means ± SE of three independent experiments; single asterisks indicate significant differences between samples, and double asterisks indicate highly significant differences between samples.