Fig. 3: JAM1 is degraded by the autophagy pathway and negatively regulates RKN resistance.

a Real-time quantitative PCR analysis of JAMs in the roots of Ailsa Craig (AC) plants at different time points after RKN infection. Total RNA was isolated from root samples collected at different time points after RKN infection. Error bars represent SD; data represent the mean ± SD (n = 3 biological replicates, individual dots). b–d Detection of JAM1 protein levels. b Changes in the protein levels of JAM1 at different time points after RKN infection. Left, western blots showing the protein levels of JAM1. Right, amounts of JAM1 determined by densitometry of protein bands from three experiments. Error bars represent SD; data represent the mean ± SD (n = 3 independent experiments, individual dots). c, d Response of WT and atg7 mutant seedlings to the ubiquitin proteasome inhibitor MG132, the vacuolar-type ATPase inhibitor Concanamycin A (ConA) and the autophagy inducer AZD8055 with or without RKN infection. Left, western blots showing the protein levels of JAM1. Right, amounts of JAM1 determined by densitometry of protein bands from three experiments, respectively. Seedlings were pretreated with MG132 (50 μM), ConA (1 μM) or AZD8055 (5 μM) for 10 h and then were analyzed by western blotting. Band intensity was quantified by ImageJ. The ratio of JAM1/Actin in the control was set to 1. The levels of JAM1 protein were determined with anti-JAM1 antibody. The Actin protein served as loading control. Error bars represent SD; data represent the mean ± SD (n = 3 independent experiments, individual dots). The experiments were repeated three times with similar results (b–d). e Phenotype of RKN reproduction in AC, jam1, jam2 and jam1/2 mutants using acid fuchsin staining after RKN infection. Bar, 1 cm. f The number of root knots of plants at 5 weeks after infection with RKNs. Data are presented as boxplots, with each dot representing the datapoint of one biological replicate (n = 25 plants). For the boxplots, the central line indicates the mean value, the bounds of the box show the 1st and 3rd quartile, and the whiskers indicate 1.5× interquartile range between the 1st and 3rd quartile. The experiments were repeated twice with similar results. g The direct fluorescence of GFP-ATG8f was detected in the roots by confocal fluorescence microscopic analysis with or without RKN infection (Mock). Bars, 25 μm. h Quantification of (g). The number of autophagosomes per image was quantified to calculate the autophagic activity relative to wild-type control plants, which was set to ‘1’. Error bars represent SD; data represent the mean ± SD (n = 20 samples, individual dots). The experiments were repeated twice with similar results. Different letters above bars indicate a significant difference at the P < 0.05 level by one-way ANOVA with Tukey’s multiple comparisons test. Exact P-values of statistic tests are provided in the Source data file.