Inhibition of ROS-Scavenging Enzyme System Is a Key Event in Tomato Genetic Resistance against Root-Knot Nematodes

Abstract

Genetic resistance in plants against incompatible pests is expressed by the activation of an immune system; however, the molecular mechanisms of pest recognition and expression of immunity, although long the object of investigation, are far from being fully understood. The immune response triggered by the infection of soil-borne parasites, such as root-knot nematodes (RKNs), to incompatible resistant tomato plants was studied and compared to the compatible response that occurred when RKNs attacked susceptible plants. In compatible interactions, the invading nematode juveniles were allowed to fully develop and reproduce, whilst that was impeded in incompatible interactions. In crude root extracts, a first assay of reactive oxygen species (ROS)-scavenging enzymatic activity was carried out at the earliest stages of tomato-RKN incompatible interaction. Membrane-bound and soluble CAT, which is the most active enzyme in hydrogen peroxide (H₂O₂) scavenging, was found to be specifically inhibited in roots of inoculated resistant plants until 5 days after inoculation, with respect to uninoculated plants. The expression of genes encoding for antioxidant enzymes, such as CAT and glutathione peroxidase (GPX), was not always inhibited in roots of nematode-infected resistant tomato. Therefore, the biochemical mechanisms of CAT inhibition were further investigated. Two CAT isozymes were characterized by size exclusion HPLC as a tetrameric form with a molecular weight of 220,000 dalton and its subunits (55,000 dalton). Fractions containing such isozymes were tested by their sensitivity to both salicylic acid (SA) and H₂O₂. It was evidenced that elevated concentrations of both chemicals led to a partial inactivation of CAT. Elevated concentrations of H₂O₂ in incompatible interactions have been suggested to be produced by membrane-bound superoxide anion generating, SOD, and isoperoxidase-enhanced activities. Such partial inactivation of CAT has been depicted as one of the earliest key metabolic events, which is specifically associated with tomato immunity to RKNs. Enhanced ROS production and the inhibition of ROS-scavenging systems have been considered to trigger all the metabolic events leading to cell death and tissue necrosis developed around the head of the invading juveniles by which this special type of plant resistance is exerted.

Keywords: RBOHD; ROS; antioxidants; catalase; hydrogen peroxide; pest resistance; root-knot nematodes; superoxide anions.

Figure 1

Numbers of egg masses per root system (EMs/RS) in tomato resistant (Res) and susceptible (Susc) plants inoculated with an avirulent (MifieldV) and a lab-selected virulent (SM2V) M. incognita population.

Figure 2

SOD, CAT, and APX activities in root crude extracts from control and resistant plants inoculated with MifieldV. Crude extracts were collected from plants harvested 1 and 2 days after inoculation (dpi). Means of enzyme activities are expressed as Units × mg⁻¹ prot ± standard deviation (SD) and separated by a paired t-test (* p < 0.01).

Figure 3

Expression of CAT, GPX, and PR4b genes in roots of tomato plants from the resistant cvsMotelle and VFN8 and the susceptible cv Moneymaker detected by quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Relative fold changes (the value 1 indicates no change) of nematode inoculated with respect to uninoculated plants were measured at 5 dpi. Data are the mean fold changes (n= 6) ± SD in gene transcript levels. An asterisk indicates that the mean fold change is significantly different from 1 as determined by the non-parametric Kolmogorov–Smirnov test (p < 0.05).

Figure 4

CAT in root extracts of tomato plants from near-isogenic lines resistant (Res, cv. Motelle) and susceptible (Sus, cv. Moneymaker) untreated or treated with SA (20 mg/plant); data were taken at 5 dpt. Values are expressed as means (n = 9) ± SD. An asterisk indicates significant difference with respect to controls (Res, Sus), according to a paired t-test (p < 0.05).

Figure 5

CAT purification from 22% ammonium sulphate precipitates of root cytosolic fractions isolated from resistant tomato cv. Motelle. Precipitates were loaded on a size exclusion column inserted in an automated HPLC system. After void volumes (up to 9th fraction), 1 mL fractions of eluate were collected until they showed detectable CAT, from 10th to 18th fraction. Absorbance of such fractions was measured at 280 and 407 nm, and expressed as arbitrary units (AU) × 100. CAT was expressed as units mL⁻¹.

Figure 6

High- (A) and low- (B) molecular-weight isoCATs (hmwtCAT and lmwtCAT, respectively) activity of AS-precipitates from root soluble fractions of tomato plants resistant (Motelle, Res) and susceptible (Moneymaker, Sus) to RKNs. CAT was assayed in absence and presence of increasing concentration of K-salicylate (0.1–1.0 mM, pH 6.0). Values are expressed as means (n = 3) ± SD.

Figure 7

HmwtCATs measured as units mg⁻¹ prot from resistant (Motelle, Res, (A)) and susceptible (Moneymaker, Sus, (B)) tomato roots at increasing H₂O₂ concentrations in presence of 0.2 mM K-salicylate (SA). Values are expressed as means (n = 3) ± SD.

Figure 8

Catalytic cycles of CAT. (A) Two-electron equivalent transfer from Ferric enzyme (heme iron positive charges in ovals) to Compound I and vice versa; (B) One-electron reduction from Compound I to Ferric enzyme through Compound II; Compound II can be oxidized by H₂O₂ into the inactive oxy-enzyme Compound III.