Plant, fungus and insect

Tomato seeds (S. lycopersicum cv. Jinbao) were surface-sterilized by 10% H 2 O 2 for 5 min and germinated in autoclaved quartz sand after rinsing with sterilized distilled water. The 10-day-old seedlings were transplanted to pots for further experimentations. F. mosseae (Nicol. & Gerd, FM) Gerdemann & Trappe BEG 167, kindly provided by Dr. Runjin Liu at Qingdao Agricultural University (China), was used to establish CMNs between two tomato seedlings in the same pot. Mycorrhizal inocula were reproduced in pots with corn (Zea mays cv. Gaoyou-115) in autoclaved sand media42 and consisted of rhizospheric sand with root segments and hypahe (35 infective propagules/gram).

A leaf-chewing caterpillar common cutworm (S. litura, SL) was used to attack tomato plants. SL larvae were reared on a semisynthetic diet containing wheat germ43 and maintained in an insectary at 23–26°C, 16 h/8 h (day/night) and 65–70% relative humidity. Homogenously 12-hour-molted third instar larvae were used to attack tomato plants.

Chemicals

TRIzol reagent, M-MLV reverse transcriptase, Taq polymerase, RNase inhibitor and dNTPs were from TaKaRa (Shuzo Co. Ltd., Shiga, Japan), 4-morpholine-propanesulfonic acid (MOPS) and diethylpyrocarbonate (DEPC) were from AMRESCO (Solon, OH), SYBR Green Real Time PCR Master Mix was from Toyobo Life Science (TOYOBO Co. Ltd, OSAKA, Japan) and methyl jasmonate (MeJA) was from Sigma-Aldrich (St. Louis, MO, USA), respectively.

Experimental design and growth conditions

A rectangular plastic pot measuring 29 × 13 × 11 cm (length × height × width) was separated by two fine stainless steel screens (25 μm, TWP Inc. Berkeley, CA, USA) into two equal compartments (Compartment I and II), which prevent direct root contacts but allowing fungal mycelia to get through screens to establish CMNs between plants. One tomato plant in the compartment I or ΙΙ was denoted as ‘donor’ and ‘receiver’ plant, respectively. To determine the direct effects of CMNs whilst excluding possible root contacts and mycorrhization, four treatments were designed (Table 1 & Fig. S1)22: A) a ‘receiver’ plant was connected with a SL-attacked ‘donor’ plant through a CMN; B) a ‘receiver’ plant was grown near a SL-attacked ‘donor’ plant without AMF inoculation or CMNs connection between plants; C) a mycorrhizal ‘receiver’ plant was grown near a SL-attacked mycorrhizal ‘donor’ plant but being separated by a water-proof membrane to prevent root and mycelial contacts between them and D) a ‘receiver’ plant was connected with the neighbouring ‘donor’ plant by a CMN without SL feeding. For enzymatic and molecular analysis all ‘receiver’ plants in these four treatments did not receive any insect attack. In bioassays all ‘receiver’ plants were infested with SL larvae.

Each compartment was filled with 1.5 kg autoclaved soil/sand mixture (2:1). The brown loam soil was obtained from the university campus containing 2.37% organic matter, 0.21% total N, 56.2 mg/kg available P with a pH of 5.64. For mycorrhizal inoculation 100 g F. mosseae inocula were applied to the compartment I in treatments A and D before sowing, but 50 g to each compartment in treatment C. In treatment B 100 g above-mentioned corn growth media without mycorrhizal inoculation were applied to the compartment I.

Plants were grown in the same conditions as Song et al.22. Under the microscopic observation CMNs between plants over the fine steel mesh were established after 35 d of transplanting and this was also confirmed at harvest (40 days after transplanting) (Table 1). Leaves of each ‘donor’ tomato were infested with five newly-molted third instar SL larvae after 40 d of transplanting. After insect infestation both ‘receivers’ and insect-attacked ‘donors’ were covered by air-tight plastic bags for 5 d to eliminate possible volatile signal contact between plants. Leaves of ‘receivers’ were collected at 3, 6, 12, 24 and 48 h after insect feeding on ‘donors’ for real-time RT-PCR and enzymatic analyses. Root AM colonization was measured according to Mukerji et al.42.

Involvement of the JA signalling pathway

Jasmonic acid (JA) signalling pathway plays an essential role in plant responses to chewing insects24,25. Both JA biosynthesis defective mutant suppressor of prosystemin-mediated responses2 (spr2) and JA signalling defective mutant jasmonic acid–insensitive1 (jai1) tomato, which were derived from a tomato wild-type (WT, Solanum lycopersicum Castlemart) parent44, were used to determine the role of JA signalling in CMNs mediated plant communication. The experiment design was similar to that described above, but spr2 was used as a ‘donor’ in treatments E, F and G to determine if a ‘receiver’ WT tomato could ‘eavesdrop’ on defence signals from its insect-attacked JA signal defective ‘donor’ through CMNs (Table 2). The treatment C in this experiment was different from the above one since the ‘donor’ did not receive both insect and AMF inoculation, but still separated by two stainless steel screens (25 μm). The spr2 and jai1 were ‘receivers’ in treatment H and I, respectively, but the ‘donors’ were the WT tomatoes receiving both insect and AMF inoculation (Table 2).

Bioassay

To test if induced defence by CMNs communication could enhance insect resistance, bioassays were conducted to compare the weight gain of larvae fed on both ‘receiver’ and ‘donor’ tomatoes. Both CMN establishment and insect feeding on the ‘donor’ plants were the same as in the above experimental design. However, in bioassays all ‘receiver’ plants were infested with third instar SL larvae 24 h after insect feeding on the ‘donor’ plants. The larval weight fed on ‘receiver’ and ‘donor’ plants were recorded 72 h after insect inoculation. Before feeding treatment all larvae had been starved for 2 h and then weighed. Four sets of bioassays were independently carried out and four pots per treatment were set up for each set of bioassays.

Enzyme assays

Four defence-related enzymes including peroxidase (POD), polyphenol oxidase (PPO), superoxide dismutases (SOD) and lipoxygenase (LOX) were analysed. Leaf samples (0.2 g fresh weight) were ground in liquid nitrogen and homogenized in 2.0 ml ice cold 0.05 M phosphate buffer (pH 7.2 for POD, but 7.8 for PPO and SOD) containing 1% (w/v) polyvinylpyrrolidone (PVP). The supernatant after 12,000 g centrifugation for 15 min at 4°C was used for enzyme assays (Song et al., 2010). Activities of LOX, POD, PPO and SOD were spectrophotometrically determined according to Rodríguez-Rosales et al.45, Kraus & Fletcher46, Zauberman et al.47 and McCord & Fridovich48, respectively. Leaf samples for enzyme analyses were harvested from ‘receiver’ plants of three sets of independent experiments with three pots per treatment for each set of experiments.

Analysis and perception of JA

Four treatments (A, B, C and D) as described above were used to determine if an application of methyl jasmonate (MeJA) on ‘donor’ plants could increase JA production and JA signalling perception in CMN-connected ‘receiver’ plants. Forty days after transplanting ‘donor’ plants in treatment A, B and C were sprayed with 1.0 ml 1.0 mM MeJA (pH 8.0, dissolved in 50 mM sodium phosphate buffer containing 0.01% Tween 20 and adjusted by 1.0 M citric acid) or in treatment D with 1.0 ml buffer lacking MeJA. Leaves from ‘receiver’ plants were then harvested for JA analysis and RNA extraction at 0, 3, 6, 12 and 24 h after the MeJA application on ‘donor’ plants. For each treatment at each time point, four plants were sampled. JA levels were determined by gas chromatography (GC) as described by Ye et al.49. CORONATINE-INSENSITIVE1 (COI1) encoding an F-box protein that is required for JA signalling perception50 was used to detect the transfer of JA signalling from ‘donor’ plants to ‘receiver’ plants.

Real-time RT-PCR analysis

Differential expression of selected genes was verified by real-time RT-PCR using the RNA samples isolated from tomato leaves obtained from all ‘receiver’ plants. The Ubi3 gene was used as a reference gene. Total RNA from tomato leaves was extracted and isolated according to Kiefer et al.51 including a DNase (Promega, Madison, USA) treatment. First strand cDNA was synthesized from 1 μg total RNA using ImProm-II™ Reverse transcription system (Promega, Madison, USA) according to the manufacturer's instructions.

The primers for target's genes LOXD, AOC, COI1, PI-1 and PI-11 were designed by Primer 3.0 software (Applied Biosystems, http://fokker.wi.mit.edu/primer3/input.htm) based on tomato mRNA sequences deposited in the GenBank. The gene-specific primer sequences used are listed in Table S1. Real-time PCR reactions were carried out with 0.2 μl (0.15 μM) of each specific primers, 1 μl cDNA, 12.5 μl of the SYBR green master mix (Quanti Tech SYBR Green kit, Qiagen, Gmbh Hilden, Germany) and the final volume made up to 25 μl with RNase-free water. In the negative control cDNA was replaced by RNase free water. The reactions were performed on a DNA Engine Opticon 2 Continuous Fluorescence Detection System (MJ Research Inc., Waltham, MA, USA). The programme used for real-time PCR was 3 min initial denaturation at 95°C, followed by 35 cycles of denaturation for 20 s at 95°C, annealing for 20 s (LOXD: 56.9°C;AOC: 56.5°C;PI-1: 47.6°C;PI-11: 55°C;COI1: °C; Ubi3: 51.5°C and extension for 20 s at 72°C. The fluorescence signal was measured immediately after incubation for 2 s at 75°C following the extension step, which eliminated possible primer dimer detection. At the end of the cycles, melting temperatures of the PCR products was determined between 65°C and 95°C. Amplicon specificity was verified by the melting curve analysis and agarose gel electrophoresis. Transcripts of targeted genes were calculated by the Double-standard Curves method. Three biological replicates were independently carried out and three pots per treatment were set up for each biological replicate. Each leaf sample for RNA extract was collected from tomato leaves of the ‘receiver’ plants in each pot.

Statistical analysis

For each treatment three replicates were maintained in a completely randomized design. SAS 8.0 (SAS Institute, Cary, North Carolina) package for windows was used for statistical analysis. The data for root AM colonization, larva weight, enzymatic activity, JA concentrations and gene expression levels were analyzed with a one-way analysis of variance with the significant differences among means identified by the Turkey multiple range test at P < 0.05.