Mi-1-mediated resistance to Meloidogyne incognita in tomato may not rely on ethylene but hormone perception through ETR3 participates in limiting nematode infection in a susceptible host

Abstract

Root-knot nematodes, Meloidogyne spp., are important pests of tomato (Solanum lycopersicum) and resistance to the three most prevalent species of this genus, including Meloidogyne incognita, is mediated by the Mi-1 gene. Mi-1 encodes a nucleotide binding (NB) leucine-rich repeat (LRR) resistance (R) protein. Ethylene (ET) is required for the resistance mediated by a subset of NB-LRR proteins and its role in Mi-1-mediated nematode resistance has not been characterized. Infection of tomato roots with M. incognita differentially induces ET biosynthetic genes in both compatible and incompatible interactions. Analyzing the expression of members of the ET biosynthetic gene families ACC synthase (ACS) and ACC oxidase (ACO), in both compatible and incompatible interactions, shows differences in amplitude and temporal expression of both ACS and ACO genes in these two interactions. Since ET can promote both resistance and susceptibility against microbial pathogens in tomato, we investigated the role of ET in Mi-1-mediated resistance to M. incognita using both genetic and pharmacological approaches. Impairing ET biosynthesis or perception using virus-induced gene silencing (VIGS), the ET-insensitive Never ripe (Nr) mutant, or 1-methylcyclopropene (MCP) treatment, did not attenuate Mi-1-mediated resistance to M. incognita. However, Nr plants compromised in ET perception showed enhanced susceptibility to M. incognita indicating a role for ETR3 in basal resistance to root-knot nematodes.

Figure 1. Root-knot nematodes (Meloidogyne incognita) induce the expression of ethylene biosynthetic genes in tomato.

In vitro grown seedlings of near isogenic tomato cvs. Moneymaker and Motelle were infected with 100–150 second-stage juvenile root-knot-nematodes in sterile conditions. The infected root tips were sampled at 0, 12, 24 and 36 h post infection (hpi). Expression of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase genes (ACO) and ACC synthase genes (ACS) was determined by semi-quantitative RT-PCR using gene-specific primers (Table S1) in two biological replicates with similar results. PCR amplification from a single sample is presented for each time point and genotype. Amplification of the tomato ubiquitin Ubi3 gene was used as internal control. PCR cycles are indicated on the right side of the panel.

Figure 2. Silencing ACS genes in tomato does not compromise Mi-1-mediated resistance to root-knot nematodes.

Two-week-old tomato plants cvs. Moneymaker (mi/mi) and Motelle (Mi-1/Mi-1) were used in agroinfiltration of tobacco rattle virus (TRV) empty vector, and cv. Motelle was used with TRV containing a portion of Mi-1 (TRV-Mi-1) or containing 1-aminocyclopropane-1-carboxylic acid synthase (ACS) constructs (TRV-ACSI and TRV-ACSII), which were either individually or simultaneously agroinfiltrated (TRV-ACSI+II). Three weeks after agroinfiltration, plants were infected with 10,000 second-stage juvenile root-knot-nematodes and evaluated for nematodes reproduction 8 weeks later. Dots represent the number of egg masses counted on a single root system (n = 18–25). Two independent experiments were performed with similar results and data from one are presented.

Figure 3. Effect of MCP treatment on ethylene response and resistance to root-knot nematode in tomato roots.

(A) Efficiency of the 1-methylcyclopropene (MCP)-blocking of ethylene (ET) perception was assessed by monitoring the expression of E4 after induction by ET. Seven-week-old cv. Moneymaker plants (+MCP/+ET) were pre-treated with MCP, and two plants were treated daily for 18 h with 10 µl/l ET prior to harvest. Root tissues were pooled and frozen. Tissues from untreated plants (−MCP/−ET) or plants only induced by ET (−MCP/+ET) were used as control. Total RNA (25 µg) for each sample was used for RNA blot analysis. The blot was hybridized sequentially with E4 and an 18S rDNA probe used to normalize expression. (B, C) Five-week-old tomato plants cvs. Moneymaker and Motelle were treated with MCP (+MCP) or untreated (−MCP) prior root-knot nematode (RKN) infection with 3,000 second-stage juvenile. During the first 2 weeks following RKN infection, the plants (+MCP) were repeatedly treated with MCP every 2 days. RKN reproduction was evaluated 7 weeks after infection as (B) egg masses and (C) egg production. Results are presented relative to the fresh weight (FW) of roots. Error bars indicate standard error of the mean (n = 16), where bars with different letters denote significant difference at P<0.05. The bioassay was performed twice with both tomato cultivars tested and twice more with cv. Moneymaker only. In all experiments, results from the same genotypes were similar. Data from one representative experiment are presented.

Figure 4. Root-knot nematodes reproduction on tomato is affected by the Nr mutation in compatible host only.

Root-knot nematodes (RKN) reproduction was evaluated on Never ripe (Nr) mutant, wild type tomato cvs. Pearson and VFN, and the Nr introgressed line VFNxNr. Four-week-old plants were infected with 3,000 second-stage juvenile RKN. (A) Egg masses and (B) eggs production were evaluated 6 weeks after RKN infection. Results are presented relative to the fresh weight (FW) of roots. Error bars indicate standard error of the mean (n = 20–30), where bars with different letters denote significant difference at P<0.05. Two independent experiments were performed with similar results and data from one are presented.