The NahG line was a gift from Prof. Dr. Cheng-Cai Chu (Chinese Academy of Sciences, Beijing, China). The Coi1-13 line was provided by Prof. Dr. Dong-Lei Yang (Nanjing Agricultural University, Nanjing, China). The OsEin2b-RNAi line was provided by Yi-Nong Yang (Pennsylvania State University, United States), and the OsWRKY45-RNAi transgenic line was a gift from H. Takatsuji (Plant Disease Resistance Research Unit, National Institute of Agrobiological Sciences, Ibaraki, Japan); all the above four lines were all with a background of Nipponbare. The mutant hebiba and its corresponding wild-type cv. Nihonmasari were generously provided by P. Nick (Karlsruhe University, Karlsruhe, German). Cultivar Tetep and Nipponbare were maintained in the lab, which were initially obtained from rice breeders.

Rice cultivar Tetep and Nipponbare were used to detect the level of resistance/susceptibility to A. besseyi and analyze the expression of plant hormone-related genes. Rice cultivar Nipponbare was also used to be sprayed by hormones or hormone inhibitors to clarify the roles of hormones in nematode defense. Five rice mutants or transgenic lines including an SA-deficient transgenic NahG line, a JA response-deficient mutant line Coi1-13, a transgenic line OsEin2b-RNAi, which is with a silenced expression of ET signaling gene OsEin2b, an OsWRKY45-RNAi transgenic line, and a JA biosynthesis mutant hebiba were used to further obtain a detailed role of the three hormone pathways in the interaction of rice and A. besseyi.

All seeds used in this research were first soaked in hot water at 56°C for 15 min to kill the seed-borne nematodes (Fortuner and Orton Williams, 1975), then germinated on wet filter paper for 3 days at 28°C, subsequently transferred to autoclaved sand mixed with 0.2% (m/v) superabsorbent polymer (SAP) substrate (Reversat et al., 1999) in 10 ml centrifuge tubes, and further were grown at 26°C under a 16/8 h light regime. To harvest the rice grains and further count the nematodes, the seedlings were transplanted into a greenhouse plot at 25 days after inoculation (dai) with a density of 250 mixed infective stages of A. besseyi per plant and grown at approximately 28–30°C and 60–70% relative humidity (RH). Plots were 1.5 m × 2.0 m and caged with nylon nets to protect against insects. An earth levee of 25 cm height was made around each plot to maintain the water level and to isolate each plot.

Aphelenchoides besseyi was isolated from infected rice seeds of AnHui1 (O. sativa subsp. japonica) by soaking seeds in tap water for 72 h. Then, nematodes were collected and surface-sterilized with 0.1% streptomycin sulfate for 10 min, washed three times with sterile double-distilled water, and subsequently cultured on carrot discs at 25°C for 30 days as described in the study by Tulek et al. (2009).

The nematodes were then collected in tap water, and approximately 250 nematodes were inoculated to each plant by using water flotation method described by Xie et al. (2019). Briefly, hot water-treated seeds were germinated for 3 days at 28°C and planted individually into the center of a 10 ml centrifuge tube containing 4 ml SAP substrate; the tubes were capped and the plants were grown for 3 days at 26°C under a 16 h/8 h light regime. Subsequently, 2 ml of nematode suspensions in tap water containing 250 mixed infective stages of nematodes were added to each plant to submerge the growth site of the seedling. The tubes were capped for another 5 days to retain the moisture. Clean tap water without nematodes was used as the mock control.

The hormone analogs benzo-1,2,3-thiadiazole-7-carbothioic acid S-methyl ester (BTH), methyl jasmonate (MeJA), and ethephon were purchased from Sigma-Aldrich (Sigma, St. Louis, United States). Diethyldithiocarbamic acid [DIECA (Farmer et al., 1994)], a JA biosynthesis inhibitor; aminooxyacetic acid [AOA (Iwai et al., 2006; Mao et al., 2006)], an inhibitor of ET biosynthesis, and L-2-aminooxy-3-phenylpropionic acid [AOPP (Amrhein and Gödeke, 1977)], an inhibitor of phenylalanine ammonia-lyase (PAL) activity were bought from FUJIFILM Wako Pure Chemical Corporation (Neuss, Deutschland) and Sigma-Aldrich (Sigma, St. Louis, United States). Two concentrations of hormones or analogs were used in the study, i.e., MeJA (250 and 500 μM), BTH (125 and 250 μM), and ethephon (250 and 500 μM). The following concentrations of the hormone inhibitor were used in this study: DIECA (100 μM), AOA (20 mM), and AOPP (100 μM). All chemicals were dissolved in water containing 0.02% (v/v) Tween 20.

The chemical treatment was examined at two stages in Nipponbare. For the analysis of the effects of chemicals on nematode infection at the seedling stage, the prepared chemicals or water control containing 0.02% (v/v) Tween 20 were sprayed with a perfume sprayer onto the leaves of six-day-old seedlings; the nematode of mixed infective stages with a density of 250 per plant were inoculated at 24 h after spraying, and a number of nematodes were collected from above ground parts at 25 dai. For the analysis of the effects of chemicals in nematode infection at flowering stage, 250 of mixed infective stages nematodes were inoculated individually onto seven-day-old seedlings and then transplanted into a greenhouse plot at 25 dai for further growth. The prepared chemicals or water control containing 0.02% (v/v) Tween 20 were sprayed onto the leaves and panicles at day one of flowering, and the number of nematodes were collected from grains at the ripen stage.

To analyze the nematode reproduction dynamics in Nipponbare and Tetep, nematodes were collected from the above ground parts at 15 dai, tillering stage (about 30 dai), jointing stage (about 45 dai), from panicles at booting stage (about 60 dai) and flowering stage (about 70 dai), and from grains at ripen stage (about 100 dai). To confirm the effects of plant hormones on nematode infection, nematodes on mutant lines or chemical-treated plants were collected at 25 dai and grains. The white-tip symptoms were observed and recorded at tillering stage. Symptom rates (%) = The number of plants showing white-tip symptoms/the number of inoculated plants × 100%.

All the counts in this study were performed according to Xie et al. (2019). Briefly, the above ground part of seedlings or panicles was cut into approximately 1 cm long pieces, immersed in 0.01% Tween 20, placed on a shaker at 40 rpm for 24 h, and then, the water was collected and the materials were rinsed twice; all the water was collected into a counting dish, and the nematodes in water were counted under a stereomicroscope. Each experiment or treatment had three biological replicates, and each replicate contained 15 plants. For evaluation of the nematode population in grains, a total of randomly selected 100 mixed seeds from one replicate of one treatment were collected and were gently ground to separate glumes and seeds, and then the glumes and grains were soaked in tap water in a Petri dish on a shaker at 40 rpm for 8 h and washed twice. Nematode observation was performed the same as described above under a stereomicroscope. Each replicate contains five randomly mixed 100 grains; three independent biological replicates were recorded.

For quantitative real-time (qRT)-PCR, the above-ground tissues (including all the leaves and stem) of Tetep and Nipponbare were separately collected at 3, 5, 7, and 14 dai, and the panicles were collected at the flowering stage after nematode inoculation. The tissues of clean tap water inoculation were served as the corresponding control. RNA was extracted using the TRIzol reagent (Invitrogen, Breda, Netherlands), and the extracted RNA was treated with DNaseI (Thermo Fisher Scientific) to remove all contaminating DNA as follows: added 1 μl (1 U) DNase I, 1 μg extracted RNA, 1 μl of 10× reaction buffer with 25 mM MgCl₂, and DEPC-treated water into RNase-free tube to a final volume of 10 μl and then incubated at 37°C for 30 min; later, 1 μl 50 mM EDTA was added and incubated at 65°C for 10 min. The RNA concentration and purity were measured using a NanoDrop 2000 (Thermo Scientific). The first-strand cDNA synthesis was generated using RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Lithuania). The PowerUp™ SYBR™ Green Master Mix (Thermo Fisher Scientific, Lithuania) was used to perform qRT-PCR: Green Master Mix (2×) 10 μl, forward primer 1 μl (10 ng/μl), reverse primer 1 μl (10 ng/μl), cDNA 1 μl (20 ng/μl), and nuclease-free water 7 μl. qRT-PCR was performed under the following conditions: 5 min at 95°C and 45 cycles of (10 s at 95°C and 30 s at 59°C). After the PCR reaction, a melting curve was generated by gradually increasing the temperature to 95°C to test for amplicon specificity. All reactions were performed in three technical replicates on Bio-Rad CFX connect (Bio-Rad, United States) and analyzed using R version 3.3.0 (R Core Team, 2016). Each biological replicate composed of a pool of eight individual plants or panicles. The experiment was independently repeated three times. Expression data were normalized using two reference genes. Primer pairs for each target or reference gene are listed in Table 1.

The data were analyzed using the R version 3.3.0 software. Normality and equality of variances were tested with Shapiro–Wilk normality test (α = 0.05) and Bartlett test (α = 0.05), respectively. Normally distributed data were analyzed using an independent samples t-test or ANOVA followed by individual comparisons with the Tukey test (α = 0.05). Non-parametric testing was carried out with the Kruskal–Wallis test with α = 0.05. All data except qRT-PCR in the same treatment were not significantly different between replicates in this research; therefore, the data presented in all the results are the means of three replicates.

The number of nematodes per plant in Nipponbare was significantly higher than the ones in Tetep at all the stages tested (Figure 1). There were no significant differences between the number of nematodes per plant at 15 dai, tillering stage, and jointing stage either in Nipponbare or in Tetep. However, a 13- to 26-fold increase of nematode numbers was observed in Nipponbare at booting and flowering stages, and the number reached 160.0 and 310.1 per plant, respectively. The number of nematodes only slightly increased in Tetep at these two stages, with a four- to eightfold increase up to 14.2 and 24.8 nematodes per plant. Besides, there were, on average, 10.6 nematodes in 100 grains for Tetep, which was significantly lower than the average of 92.7 nematodes in 100 grains for Nipponbare.

The typical white tip symptom is the production of chlorotic tips on flag leaves starting at tillering, which may subsequently necrotize. This symptom was recorded in both Nipponbare and Tetep at the tillering stage, at a rate of 72.9% for Nipponbare, while in Tetep, no typical symptom was observed at the whole growth stages (Supplementary Figure 1).

The possible roles of plant hormones during the interactions of rice and A. besseyi were investigated by evaluating the expression levels of 13 selected SA, JA, or ET marker genes and 2 PR genes in Nipponbare and Tetep at early seedling stages (Figure 2).

For SA signaling pathway, two biosynthesis genes OsICS1 and OsPAL1, two SA response genes OsWRKY45 and OsNPR1, and one SA-inducible PR gene OsPR1b were analyzed. During the interaction of Nipponbare and A. besseyi, the expression levels of OsPAL1 and OsICS1 were consistently downregulated at all time points compared with those in mock control. However, the transcript level of OsWRKY45 was slightly increased in shoot tissues at 3 dai but was significantly upregulated at 5, 7, and 14 dai. The expression of OsNPR1 was not significantly affected by A. besseyi at the most of stages; however, the transcription level of OsNPR1 was significantly upregulated at 14 dai. The OsPR1b expression levels were downregulated at all times, especially at 3 and 14 dai. During the interaction of Tetep and A. besseyi, OsICS1, OsPAL1, OsWRKY45, and OsNPR1 showed no significant changes upon A. besseyi inoculation at all time points, while downregulation at 3 dai and upregulation at 7 and 14 dai were observed in OsPR1b.

To investigate the JA response, OsAOS2 (a key enzyme in JA biosynthesis), OsLOX3 (a JA synthesis pathway-related gene), OsJMT1 (an enzyme that converts JA to the volatile component MeJA), and OsJAmyb (a JA-inducible Myb transcription factor) were analyzed. The expression levels of OsAOS2 at 5 dai and OsJAmyb at 14 dai were significantly upregulated in Nipponbare, while the other genes had no significant changes at all time points. In contrast, during the interaction of Tetep and A. besseyi, the expression of OsJAmyb was significantly upregulated at 3, 7, and 14 dai, while all the other genes showed similar expression levels compared with control plants.

Genes such as OsACO7, OsACS1, OsEin2b, and OsERF1 that play key roles in ET biosynthesis or signaling were analyzed by using qRT-PCR. The expression of OsACO7 was significantly upregulated at 14 dai in Nipponbare but was downregulated at 5 dai in Tetep. The expression of OsACS1 was significantly downregulated in Nipponbare at 3, 5, and 7 dai, but only minor upregulation was detected in Tetep at the same stages. No significant differences were detected for OsEin2b between the inoculated and control plants. Nevertheless, ET-inducible gene OsERF1 appeared to be significantly down-regulated at 7 dai in Nipponbare, but no significant transcription alteration was detected in Tetep at these stages.

To assess the general defense responses in these two cultivars, after A. besseyi inoculation, the expressions of two PR genes OsPR1a and OsPR10 were analyzed. OsPR1a and OsPR10 were both significantly upregulated at all the time points except at 3 dai in Nipponbare. In contrast, the mRNA level of OsPR1a was dramatically downregulated at 3 dai and upregulated at 7 dai in Tetep; the expression levels of OsPR10 were significantly induced at 3 and 7 dai.

As shown in Figure 1, the number of nematodes is massively increasing at the flowering stage in rice panicles. Therefore, the expression levels of the same plant hormone marker genes in panicles were analyzed to investigate the role of plant hormones during A. besseyi infection at the flowering stage (Figure 3).

During the interaction of Nipponbare and A. besseyi, the SA marker genes were significantly downregulated at this stage compared with corresponding control. On the contrary, all these genes showed significantly upregulated expression levels in Tetep upon A. besseyi infection at this time points compared with those in the control.

For the key genes involved in JA response, the expressions of OsAOS2 and OsJAmyb were down-regulated, while OsLOX3 was upregulated at the flowering stage in Nipponbare. In contrast, in Tetep, only the expression of OsAOS2 was upregulated by A. besseyi infection, and all the other genes showed similar expression level compared with the control.

For ET signaling marker genes, OsERF1 appeared to be significantly upregulated both in Nipponbare and Tetep. OsACO7 was significantly upregulated in Nipponbare, but this gene was not affected by A. besseyi infection in Tetep. Moreover, the other two genes showed no significant differences in the transcription level in both cultivars compared with the corresponding controls.

OsPR1a and OsPR10 were not significantly affected in Nipponbare but were both significantly downregulated in Tetep at the flowering stage after nematode infection.

To analyze the role of SA, JA, and ET in rice defense against A. besseyi, the number of nematodes was examined at two stages in Nipponbare. The results indicated that both in plants of 25 dai and in grains, the number of A. besseyi significantly reduced in hormone-treated plants when compared with the corresponding control in two concentrations (Figure 4 and Supplementary Figure 2), and the results showed the two concentrations had similar effects on nematode infection. As the results showed in Figure 4, in BTH-, MeJA-, and ethephon-treated plants, the number of nematodes exhibited decrease of 79.91, 77.58, and 71.47% at 25 dai, respectively. While in grains, BTH-, MeJA-, and ethephon-treated plants showed 63.71, 73.96, and 70.39% nematode reductions, respectively.

In contrast, chemical inhibition of the phenylpropanoid pathway with AOPP, JA biosynthesis with DIECA, or ET production with AOA all resulted in a significantly increased susceptibility toward A. besseyi both in 25 dai plants and grains (Figure 4). In 25 dai plants and grains, compared with those in control, AOPP-, DIECA-, and AOA-treated plants showed an increase of 91.51 and 19.76%, 83.84 and 50.78%, and 16.43 and 39.70% nematodes, respectively.

To obtain a more detailed understanding of the role of these defense hormone pathways against A. besseyi, the number of nematodes was evaluated in the mutants or transgenic lines impaired in hormone pathways. Both in 25 dai plants and grains, the SA-signaling deficient mutant WRKY45-RNAi, the SA-deficient mutant NahG, and the JA insensitive mutant Coi1-13 all showed a significant increase in the number of nematodes compared with the corresponding control of Nipponbare. Nevertheless, the ET-insensitive mutant EIN2b-RNAi did not have a significant change in the number of nematodes compared with that in control either in 25 dai plants or grains. The nematode number in the JA biosynthesis mutant hebiba had no significant change in 25 dai plants but was significantly increased in grains in comparison with control Nihonmasari (Figure 5).