The Potato StNAC2-StSABP2 Module Enhanced Resistance to Phytophthora infestans Through Activating the Salicylic Acid Pathway

Abstract

Potato late blight is an important disease in potato production, which causes serious damage. Salicylic acid (SA) is a plant hormone involved in the regulation of potato (Solanum tuberosum) resistance to Phytophthora infestans. In this study, it was found that exogenous methyl salicylate (MeSA) treatment could significantly enhance the resistance of potato to P. infestans. RNA-seq results confirmed that SA was important for potato resistance to P. infestans. Salicylic acid binding protein 2 (SABP2) is a member of α/β hydrolase family, which can convert MeSA into SA to regulate the steady state of SA in plants. StSABP2 protein was obtained through prokaryotic expression, and enzymatic analysis in vitro confirmed that StSABP2 could transform MeSA into SA. In order to explore the function of StSABP2 in the process of plant resistance to P. infestans, we carried out virus-mediated gene silencing of StSABP2 in potato and transiently expressed StSABP2 in tobacco. The results showed that StSABP2 positively regulated plant resistance to P. infestans, and this process was achieved by mediating the transcription of SA signal and defence-related genes. Then we screened for the upstream regulator of StSABP2. The results of double luciferase and yeast one-hybrid analysis showed that StNAC2 could activate the transcription of StSABP2. The StNAC2-StSABP2 module regulated potato resistance to P. infestans by positively mediating the SA pathway. This study provides a new idea for improving host resistance to potato late blight by regulating the SA signal in potato and provides germplasm resources for potato resistance breeding.

Keywords: Phytophthora infestans; StNAC2; StSABP2; potato; salicylic acid.

FIGURE 1

Exogenous methyl salicylate (MeSA) enhanced potato tolerance to Phytophthora infestans. (a and b) After 120 h of infection by P. infestans , the diameter of the lesion in the control group leaves (a) and MeSA treatment group leaves (b) were observed. The leaves were taken from below the third compound leaf of potato plants previously sprayed with 10 mL of 5 mM MeSA on three successive days. The experiments were repeated three times, with similar results. (c) Lesion diameter of control and MeSA‐treated plants. Significant difference by t test, **p < 0.01.

FIGURE 2

RNA‐seq revealed the key pathway by which methyl salicylate (MeSA) imparts resistance to late blight in potato. (a) Results from GO term analysis. The number of significantly enriched differentially expressed genes (DEGs) within GO divisions is shown. The colour gradient, ranging from blue to red represents low, middle and high p over the course of the storage period. (b) Results from KEGG enrichment analysis for DEGs. The colour gradient indicates different p. (c) Effect of MeSA treatment on the involvement of salicylic acid (SA) and jasmonic acid (JA) transduction on plants. Red and blue colours represent up‐regulation and down‐regulation of genes, respectively.

FIGURE 3

Expression pattern and kinetic characterisation of StSABP2. (a) Changes in StSABP2 transcript levels at different times after infection with Phytophthora infestans . Significant difference by t test, **p < 0.01. (b) SDS‐PAGE of purified His‐tagged StSABP2 protein, with bands in the range of 25–33 kDa, indicating successful purification. Samples added to each lane of SDS‐PAGE, 1–3: Purified StSABP2 protein; 4: The supernatant after ultrasonic disruption of cells; 5–6: The liquid that flows out after the supernatant passes through the nickel column; 7: Precipitation after ultrasonic disruption of cells. (c–e) StSABP2 substrate specificity. The biochemical activities of recombinant purified StSABP2 were determined with 0, 400, 800, 1200, 1600, 2000, 3000 and 4000 μM methyl salicylate (MeSA), methyl jasmonate (MeJA) and methyl indoleacetic acid (MeIAA), and after 30 min of reaction, the contents of SA, JA and IAA in the reaction mixture were determined. The line indicates the nonlinear least‐squares fit of the initial velocity versus MeSA (c), MeJA (d) or MeIAA (e) concentration to the Michaelis–Menten equation.

FIGURE 4

Silencing of StSABP2 reduced the tolerance of potato plants to Phytophthora infestans. (a) The relative expression of StSABP2 in silenced lines. Relative expression of StSABP2 in gene‐silenced plants (TRV‐BP2‐1 and TRV‐BP2‐2) was determined by reverse transcription‐quantitative PCR compared to the control (TRV). (b and c) Measurement of disease symptoms and spot diameter of StSABP2‐silenced plants and controls 120 h after infection by P. infestans . The experiments were repeated three times, with similar results. (d–f) Changes of salicylic acid (SA) (d), jasmonic acid (JA) (e) and methyl salicylate (MeSA) (f) contents in control and silent lines at 0 h and 120 h after infection with P. infestans . (g–i) The relative expression levels of systemic acquired resistance (SAR)‐related genes StPR1 (g), StNPR1 (h) and StPR5 (i) in control and silenced plants. (j–l) Relative expression of SA synthesis genes StPAL (j), StICS1 (k) and JA signalling pathway‐related gene StMYC2 (l) in control and silenced plants. Significant difference by t test, *p < 0.05, **p < 0.01.

FIGURE 5

The transient expression of StSABP2 in Nicotiana benthamiana enhanced the plant's resistance to Phytophthora infestans . (a) 3301‐GUS and 3301‐StSABP2 were agroinfiltrated into N. benthamiana leaves. Three days after infiltration, the transcription level of StSABP2 was measured. (b) Incidence of disease 5 days after inoculation with P. infestans at the infiltration site. Representative images taken under normal light (left) or blue light (right) show lesion development. (c) The diameter of the lesion and the average lesion area. The experiments were repeated three times, with similar results. (d–g) StSABP2 promotes the expression of systemic acquired resistance (SAR)‐related genes NbPR1 (d), NbPR2 (e), NbPR5 (f) and NbNPR1 (g). (h–j) Changes of salicylic acid (SA) (h), jasmonic acid (JA) (i) and methyl salicylate (MeSA) (j) contents in 3301‐GUS and 3301‐StSABP2 at 0 and 120 h after infection with P. infestans . 3301‐GUS and 3301‐StSABP2. (k–n) Changes in relative expression of SA synthesis genes NbPAL (k) and NbICS1 (l) and JA signalling pathway‐related gene NbMYC2 (m) and NbCOI1 (n) in 3301‐GUS and 3301‐StSABP2. Significant difference by t test, *p < 0.05, **p < 0.01.

FIGURE 6

StNAC2 activated transcription of StSABP2 to enhance plant resistance to Phytophthora infestans . (a and b) The dual luciferase assay shows that StNAC2 interacted with StSABP2 and could activate the transcription of StSABP2. LUC:StSABP2 + 3301:GUS were used as a control, leaf samples were analysed, and luminescence imaging was performed 3 days after infiltration (a). Relative luciferase activity was measured using a microtitre plate reader (b). Significant difference by t test, **p < 0.01. (c and d) The yeast one‐hybrid assay shows that StNAC2 and StSABP2 interact. pHIS2‐StSABP2 + pGADT7‐StNAC2 and pHIS2‐StSABP2 + pGADT7‐empty grew normally on SD/−Leu/−Trp (c), and their growth is comparable. pHIS2‐StSABP2 + pGADT7‐StNAC2 showed significantly better growth than pHIS2‐StSABP2 + pGADT7‐empty on SD/−Leu/−Trp/−His with 3‐AT (d).