4.4.2. Establishment of Standard Curve

The plasmid solutions of s containing the right size of DNA fragments were amplified again using plasmids primer RV-M/M13-47 by qRT-PCR (BIO-RAD CFX96, USA) (20-µL reaction contained 0.4 µM each primer, 0.5 U SYBR Premix EX Taq II (2×), cDNA and nuclease-free water following the Takara RR820A instructions; 95 ◦C for 2 min, 95 ◦C for 5 s, 55 ◦C to anneal for 30 s, 72 ◦C for 30 s, for 30 cycles). The DNA concentration of target genes were detected by a NanoDrop2000 spectrophotometer (Thermo Scientifi, USA) and then diluted to 0, 1, 2, 3, 4 ng/µL. The standard curves were drawn based on the DNA concentration of target genes and Ct values (Supplementary Figure S2). The calculation of plasmid copy number was based on Shirima et al. (2017) [72].

$$\text{Plasmit copy number} = \frac{6.02 \times 10^{23} \times \left(\text{copies-mol}^{-1}\right) \times \text{plasma amount} (\text{g}) \times}{\text{MW}} \tag{1}$$

MW <sup>=</sup> plasmid molecular weight, (=plasmid size (2692 bp) <sup>×</sup> molar mass per base (660 g·mol−<sup>1</sup> ·bp−<sup>1</sup> ); 6.02 <sup>×</sup> <sup>10</sup><sup>23</sup> molecules/mole <sup>=</sup> Avogadro's constant; \* Plasmid amount was calculated from the plasmid concentration determined by a NanoDrop2000 spectrophotometer (Thermo Scientifi, USA).

#### 4.4.3. Determination of AQ-PCR

Ct values of recombinant plasmid containing 5 gradient concentration (as control) and soil DNA extracts were detected using specific primers of PS and FO by qRT-PCR (20-µL reaction was as mentioned above). The copy numbers of PS and FO were calculated based on the standard curve. All reactions were replicated three times.

#### *4.5. qRT-PCR Analysis of NB-LRRs*

For 35 previously identified *NB-LRRs* (Supplementary Table S2), RNA extraction of roots, reverse transcription and qRT-PCR analysis (BIO-RAD CFX96, USA) were conducted as described by Chen et al. (2018) [41]. All reactions were replicated three times. The data were normalized on the basis of the 18S rRNA threshold cycle (Ct) value. The samples with the NP treatments were used as the controls at the same sampling time, and their normalized Ct values were set to 1. The relative gene expression of the other treatments was calculated using the 2−∆∆CT method [73].

#### *4.6. Measurement of ABA, SA, ET and JA*

The contents of abscisic acid (ABA), salicylic acid (SA), ethylene (ET) and jasmonate (JA) were determined using a one-step double-antibody sandwich enzyme-linked immunosorbent assay (ELISA). Briefly, 1.0 g of fresh root was ground in 5 mL of phosphate-buffered saline (PBS) (0.01 M, pH 7.4) with an ice-cooled mortar and centrifuged at 2500 rpm for 20 min at 4 ◦C to obtain a supernatant for the ELISA analysis following the protocol described in Zhao et al. (2006) [74]. The mouse monoclonal antigen and antibodies against free ABA, SA, ET and JA were provided by MLBIO Co. Ltd., Shanghai, China. The hormone content was measured at 450 nm using a microplate reader (BIO-Tek ELX800, USA). Calculations of the ELISA data were performed as described in Wang et al. (2012) [75]. The recovery percentages obtained by using internal standards during extraction and analysis were all >90%.

#### *4.7. Measurement of Root Activity and the Physiological Index*

To determine root activity, the methodology described by Zhang et al. (2013) [76] was followed with modifications. Approximately 0.5 g of fresh root was mixed with 10 mL of a half-and-half blend of 0.4% triphenyl tetrazolium chloride (TTC) and 1/15 M PBS (pH 7.4) and incubated at 37 ◦C for 1 h, and then the reaction was stopped by 2 mL of 1 M H2SO4. The roots were homogenized in ethyl acetate with a capacity of 10 mL. The absorbance of the final solution was measured at 415 nm (Pgeneral T6-1650E, China). A standard curve was used to determine the concentration of root activity in the extract.

The determination of SOD, POD and CAT activities and MDA content were as described by Li et al. (2017) [48] and Deenamo et al. (2018) [77], respectively. The pretreatment was the same, and then 0.5 g of roots was homogenized in 5 mL of precooled PBS (0.05 M, pH 7.8) with a small amount of quartz sand. Extracts were centrifuged for 15 min at 13 000 rpm. The supernatant was used for the measurement of the four indexes. The colorimetric wavelengths were 560 nm, 470 nm and 240 nm for SOD, POD and CAT, and 600 nm, 532 nm, 450 nm for MDA (Pgeneral T6-1650E, China).

The H2O<sup>2</sup> content was determined by the KI method [78]. In short, 0.2 g of roots was homogenized in 0.8 mL of precooled 0.1% TCA (trichloroacetic acid) with liquid nitrogen. Extracts were centrifuged for 20 min at 19,000 rpm. Then, 0.5 mL of supernatant was added to 2 mL of KI (1 M) and 0.5 mL of PBS (100 M) for reaction at darkness for 1 h. The absorbance of the final solution was measured at 390 nm (Pgeneral T6-1650E, China). A standard curve was used to determine the concentration of H2O<sup>2</sup> in the extract.
