Plant secondary metabolite citral interferes with Phytophthora capsici virulence by manipulating the expression of effector genes

Abstract

Phytophthora capsici is a notorious pathogen that infects various economically important plants and causes serious threats to agriculture worldwide. Plants deploy a variety of plant secondary metabolites to fend off pathogen attacks, but the molecular mechanisms are largely unknown. In this study, we screened 11 plant secondary metabolites to evaluate their biofumigation effects against P. capsici, and found that citral, carvacrol, and trans-2-decenal exhibited strong antimicrobial effects. Intriguingly, a low concentration of citral was effective in restricting P. capsici infection in Nicotiana benthamiana, but it was unable to inhibit the mycelial growth. A high concentration of citral affected the mycelial growth and morphology, zoospore germination, and cell membrane permeability of P. capsici. Further investigations showed that citral did not induce expression of tested plant immunity-related genes and reactive oxygen species (ROS) production, suggesting that a low concentration of citral could not trigger plant immunity. Moreover, RNA-Seq analysis showed that citral treatment regulated the expression of some P. capsici effector genes such as RxLR genes and P. cactorum-fragaria (PCF)/small cysteine-rich (SCR)74-like genes during the infection process, which was also verified by reverse transcription-quantitative PCR assay. Five candidate effector genes suppressed by citral significantly facilitated P. capsici infection in N. benthamiana or inhibited ROS triggered by flg22, suggesting that they were virulence factors of P. capsici. Together, our results revealed that plant-derived citral exhibited excellent inhibitory efficacy against P. capsici by suppressing vegetative growth and manipulating expression of effector genes, which provides a promising application of citral for controlling Phytophthora blight.

Keywords: Phytophthora blight; antimicrobial activity; mechanism; plant secondary metabolite; virulence gene.

FIGURE 1

Comparison of the antimicrobial activities of 11 plant secondary metabolites (PSMs) against Phytophthora capsici in vitro. The 11 PSMs exhibited different inhibitory activities on P. capsici colony morphology (a) and inhibition rate (b). The colony was cultured on V8 medium at 25°C, followed by fumigation with 25 μg/mL of each PSM for 2 days. An equal volume of ethanol was used as a control (CK). Bars represent SE for three independent biological experiments.

FIGURE 2

The minimum inhibitory concentrations (MICs) of citral, carvacrol, and trans‐2‐decenal to Phytophthora capsici. Each plant secondary metabolite was dissolved in ethanol to obtain a series of diluted concentrations. P. capsici was exposed to different concentrations of citral (a), carvacrol (b), and trans‐2‐decenal (c) for 3 days, and colony morphology, colony diameter, and inhibition rate were evaluated. An equal volume of ethanol was used as a control (CK). The experiment was repeated three times with similar results.

FIGURE 3

A low concentration of citral directly inhibited Phytophthora capsici infection in Nicotiana benthamiana. (a) Disease symptoms on N. benthamiana leaves after fumigation with low concentrations of each plant secondary metabolite (PSM). Detached N. benthamiana leaves were inoculated with P. capsici, and a low concentration of each PSM was placed adjacent to the leaves. An equal volume of ethanol was used as a control (CK). After 36 h, the inoculated leaves were photographed under UV light. (b) The lesion area of inoculated leaves after fumigation with low concentration of each PSM for 36 h. The lesion area was calculated from three independent biological replicates with at least eight leaves per replicate. (c) Relative biomass of P. capsici in the inoculated leaves after fumigation with each PSM. Infected leaves were collected at 36 h after inoculation for DNA isolation and quantitative PCR analysis. (d) Methodology to test the inhibition effects of citral on P. capsici‐infected N. benthamiana plants. (e) Representative photographs showed that a low concentration of citral could inhibit P. capsici infection in N. benthamiana. Photographs were taken at 36 h after inoculation with P. capsici zoospores. The black‐brown sites showed the infected area, and the bright red areas showed the healthy parts. This assay included three replicates, and each replicate included five N. benthamiana plants. (f) Relative biomass of P. capsici in the inoculated N. benthamiana plants after fumigation with 1.4 μg/mL citral. Asterisks indicate a significant difference according to Student's t test; **p < 0.01.

FIGURE 4

Citral affected the mycelial morphology, zoospore germination, and cell membrane permeability of Phytophthora capsici. (a) Effects of citral on the mycelial morphology of P. capsici. After fumigation with 1.4 and 22.9 μg/mL citral for 3 days, the mycelial morphology was observed and photographed under a light microscope. (b) Effects of citral on zoospore germination of P. capsici. An equal number of zoospores was cultured on V8 medium containing 1.4, 11.5, and 22.9 μg/mL citral. The germination rate of zoospores was recorded at 3, 6, and 12 h. (c) The effects of citral on malondialdehyde (MDA) content of P. capsici. After treatment with citral for 48 h, the mycelia of P. capsici were harvested to detect the MDA content using an MDA detection kit. The assays were performed three times. Asterisks indicate a significant difference according to Student's t test; **p < .01. CK, control (ethanol) treatment.

FIGURE 5

A low concentration of citral did not contribute to plant immunity. (a) Relative expression of four plant defence‐related genes after treatment with citral at different times. The transcript levels of the genes were measured by reverse transcription‐quantitative PCR, and NbActin was used to normalize relative expression. This experiment was repeated three times with similar results. (b) Production of reactive oxygen species in Nicotiana benthamiana treated with 1 μM flg22 or 1.4 μg/mL citral. This assay was repeated three times with similar results. CK, control (ethanol) treatment.

FIGURE 6

Citral suppressed the expression of Phytophthora capsici virulence genes based on RNA‐Seq analysis. (a) Statistics of up‐regulated and down‐regulated P. capsici genes between citral treatment and the control. (b) Statistics of differentially expressed genes encoding secreted proteins. On the right‐hand side of the figure, various groups of differentially expressed genes (DEGs) encoding virulence proteins are displayed. (c) Validation of RNA‐Seq data by reverse transcription‐quantitative PCR analysis. The PcActin gene was used as the internal control. This assay was repeated three times. Asterisks indicate a significant difference according to Student's t test; **p < 0.01. CK, control (ethanol) treatment.

FIGURE 7

Candidate genes whose expression was inhibited by citral contributed to Phytophthora capsici virulence. (a) Five proteins neither induced cell death nor inhibited the cell death triggered by INF1. Each of the five proteins was infiltrated first, and then INF1 was infiltrated after 24 h. The leaves were photographed after infiltration for 4 days. The numbers in the figure represent different proteins: 1, GFP; 2, PcAvh254; 3, PcAvh214; 4, PcAvh478; 5, PcSCR2; 6, PcSCR1; 7, Al106 + INF1; 8, GFP + INF1; 9, PcAvh254 + INF1; 10, PcAvh214 + INF1; 11, PcAvh478 + INF1; 12, PcSCR2 + INF1; 13, PcSCR1 + INF1. Al106 is an effector from Apolygus lucorum that can inhibit the cell death caused by INF1 and was used as a positive control. This assay was repeated three times. Asterisks indicate a significant difference according to Student's t test; **p < 0.01, *p < 0.05. (b) Immunoblot analysis of five proteins. The asterisks represent the position of the target protein. The anti‐HA antibody was used to detect the expression of the protein. Western blot showed that all the proteins were expressed normally. Ponceau S staining of the RuBisCO protein was used as the equal loading control. (c) Production of reactive oxygen species (ROS) excited by flg22. ROS accumulation was recorded over 30 min. (d) Transient expression of five candidate proteins in Nicotiana benthamiana promoted plant susceptibility to P. capsici. The infiltrated area expressing green fluorescent protein (GFP) or each protein was inoculated with P. capsici. At 36 h postinoculation (hpi), the lesion area was calculated from one experiment that contained eight leaves. This assay was repeated three times with similar results. (e) The lesion area of inoculated leaves expressing each protein. (f) The relative biomass of P. capsici in the inoculated leaves expressing each protein. Infected leaves were collected at 36 hpi and used for DNA extraction and quantitative PCR analysis.