Experimental design
The present study consisted of two main experimental parts. First, we used metabolomics and transcriptome technology to compare the metabolite differences between the EF and EI leaves that were either inoculated with the pathogen or not. We discovered that the JA, ET, and Pip were involved in endophyte-associated disease resistance in the host. Afterward, exogenous phytohormone treatments were used to verify the effects of plant hormones.
Metabolomics and transcriptome experiment: two factors were considered under a randomized block design. The first factor was the status of the endophyte (EI or EF). The second factor was pathogen inoculation (control or inoculated). Each treatment was replicated 9 times, with two pots as one replicate; a total of 72 pots were prepared. After 90 days of EF and EI plants cultivation, a spore suspension of C. lunatawas uniformly sprayed on leaves until the liquid dripped off, and the control leaves were sprayed with sterile water containing 0.02% tween 20. On the fifth day post pathogen inoculation, 24 pots of each EF and EI plant were sampled for metabolite extraction and phytohormones determination, while 12 pots of each EF and EI plant were sampled for RNA extraction: the second and third leaves from the newborn leaf in each tiller were collected and frozen in liquid nitrogen and stored at -80 oC. The experiment began in August 2020 and was conducted in the campus experimental field at Nankai University, Tianjin, China. During the experiment, the position of the pots was randomly changed every week to eliminate the position effect. Each pot was watered and fertilized as needed.
Verification experiment: The experiment was carried out on 110-old days EF and EI plants. A total of 2 × 5 treatments were applied (control, MeJA, ETH, SA, or Pip). Five biological replicates were set for each treatment, resulting in a total of 50 pots. The method of screening optimum concentrations are described in Method S1. A 25 mL aliquot of the solutions (containing 0.5% (V/V) dimethyl sulfoxide (DMSO)) of MeJA (0.2 mM), ETH (0.2 mM), SA (0.2 mM), and Pip (1mM) was sprayed on each plant. Ultrapure water containing 0.5% (V/V) DMSO was used as a control (Fig. S1). After 24 hours, we performed pathogenic inoculation as described above. Five days later, the disease index (DI) was calculated.