Plant materials
Two sets of N. benthamiana plants were used: (a) wild-type (seeds were obtained from Key Laboratory of Tobacco Pest Monitoring, Controlling & Integrated Management, Tobacco Research Institute), (b) NbMLP28 overexpressing transgenic plants (constructed in current study) were grown in a growth chamber with 50–60% humidity and a 16 h/8 h light/dark photoperiod at 25 °C. For inoculation, PVY-GFP (obtained from Key Laboratory of Tobacco Pest Monitoring, Controlling & Integrated Management, Tobacco Research Institute) was used in this study.
Cloning and sequence analysis of NbMLP28
RNA was isolated from N. benthamiana leaves using the TRIzol reagent (Vazyme) and first-strand cDNA synthesis was carried out using 2 μg total RNA and 100 U reverse transcriptase (Vazyme). Gene-specific primers MLP28F and MLP28R (Supplementary Table 1) were designed based on N. benthamiana genome data of Sol Genomics Network and used for PCR amplification; the resulting amplicons were subjected to 1% agarose gel electrophoresis and Sanger-sequenced. The deduced amino acid sequences of MLP28 (designated NbMLP28) were aligned with orthologs in other species in DNAMAN and SWISS-MODEL was employed for structure prediction [37]. The phylogenetic tree was generated using MEGA7 [38]. The potential cis-regulatory elements within NbMLP28 promoter were analyzed using the online program Plant CARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).
Virus-induced gene silencing (VIGS)
TRV vectors were kindly provided by Dr. Yule Liu, Tsinghua University, Beijing, China. Preparation of the pTRV vectors and Agrobacterium tumefaciens for VIGS followed a previously described procedure [39]. For VIGS vector construction, a 200 bp partial coding sequence (CDS) of NbMLP28 was amplified from a cDNA library of N. benthamiana leaf using gene-specific primers MLP28-TRVF and MLP28-TRVR (Supplementary Table 1) and inserted into the pTRV2 vector. For the VIGS assay, pTRV1 or pTRV2 constructs harboring the NbMLP28 fragment were introduced into the Agrobacterium strain LBA4404. Equal amount of Agrobacterium cultures containing pTRV1 and pTRV2 or pTRV2-MLP28 was mixed and used to inoculate the lower leaves of four-leaf stage N. benthamiana plants using a 1-mL needleless syringe. To determine VIGS efficiency, the leaves of tobacco plants 14 days post-inoculation (dpi) were tested by qRT-PCR using primers MLP28 QF and MLP28 QR (Supplementary Table 1), which detected the sequence outside the targeting fragment on the pTRV2-MLP28. Positive silencing plants were selected 14 dpi for analyzing NbMLP28 function. To test the response of NbMLP28 to hormones, we employed the same method described above to silence key hormone signaling genes NPR1, COI1 and EIN2 from the SA, JA and ET signaling pathways, respectively, and compared the expression levels of NbMLP28 and PVY-GFP in N. benthamiana.
Vector construction and agrobacterium-mediated gene transformation
To overexpress NbMLP28, the CDS of NbMLP28 was amplified from N. benthamiana with primers MLP28-35SF and MLP28-35SR, which contain the XbaI and EcoRI restriction sites, respectively (Supplementary Table 1). The resulting PCR fragment was inserted between the restriction sites on the Fu46-RFP entry vector. The target fragment was then inserted into the pEarlyGate100 expression vector that contains the 35S promoter, the resulting construct was introduced into the A. tumefaciens strain LBA4404 using a freeze–thaw method. A. tumefaciens cultures carrying 35S::MLP28::RFP were incubated overnight at 28 °C, harvested the next morning, and resuspended and cultured in an infiltration buffer containing 10 mM MES (pH = 5.6), 10 mM MgCl2, and 150 μM acetosyringone until OD600 reached 0.8. After three-hour incubation at room temperature, the bacterial suspensions were used to infiltrate the lower leaves of N. benthamiana plants using a needleless syringe for transient overexpression experiments.
We also overexpressed NbMLP28 in wild-type N. benthamiana using the same overexpression construct. First, a 5–8 mm disc was taken from a sterile tobacco leaf using a puncher, and the disc was placed on a preculture medium, and cultured at 25 °C for 24 h under light for 18 h. Then, the Agrobacterium of the vector was suspended in a liquid co-cultivation medium, the OD value was adjusted to 0.5–1.0, and the explants were inoculated for 30 min. The explants were placed on the co-culture medium and cultured at 24 °C for 3 days under light for 18 days. After the completion of the co-cultivation, the explants were transferred to a selection medium, cultured at 28 °C, 18 h light, and subcultured once every 2 weeks. In the selection medium, the explants grew longer and the buds grew from the callus. When the bud point grows to a length of 3 mm, it was transferred to the rooting medium. The tobacco plants were moved to the culture soil after about 2 weeks. Positive seedlings were detected with primers E100F/E100R, and the seeds were subcultured. We disinfected the surface of the T3 seeds and placed it on one-half MS medium of 50 mg/L Kan. After 1 week, the seedlings were all green, indicating that homozygous transgenic seeds had been obtained. In addition, tobacco leaves that overexpress NbMLP28 were infected with PVY-GFP after confirming the expression of 35S::MLP28 by PCR and western blotting analysis.
GFP and RFP imaging
The subcellular localization of NbMLP28 was examined using a Leica SP8 confocal microscope (Leica Microsystems, Shanghai) 48 h after the transient expression of NbMLP28 with a RFP tag in N. benthamiana epidermal cells. The plants were grown under a 16 h/8 h light/dark cycle at 25 °C. For the subcellular localization experiment, GFP was excited with a 25 mW, 488 nm argon laser, and emitted light with a wavelength between 495 and 535 nm was captured; RFP was excited with a 25 mW, 552 nm argon laser, and emitted light with a wavelength between 580 and 630 nm was captured. Successive images of 20 μm × 20 μm were scanned sequentially using 488 nm and 552 nm lasers with a 1.0 s scanning interval [40]. For the NbMLP28 silencing and overexpression experiments, in order to visually detect the accumulation of virus in inoculated leaves, we infiltrated the tobacco leaves with PVY-GFP and observed the difference in fluorescence between the treated and the control under a hand-held UV lamp (Ultra-Violet Products, Upland, CA, USA). One inoculated leaf per plant was measured and three biological replicates were analyzed for each line.
Hormone treatment
Four-week-old wild-type N. benthamiana seedlings were grown in a growth chamber under conditions mentioned above. The leaves were sprayed with 0.5 mM SA, 0.1 mM Me-JA, or 0.05 mM Ethephon with 0.02% Tween 20. The control plants were sprayed with water and 0.02% Tween 20. Three biological replicates of the wild-type N. benthamiana were analyzed. The treated leaves were harvested 24 h after the treatments, immediately snap frozen in liquid nitrogen and stored at − 80 °C until use.
Quantitative real-time PCR
Total RNAs isolation and cDNA synthesis followed the same procedures described above. qRT-PCR was performed with the SYBR Premix Ex Taq™ kit (Vazyme) using the Applied Biosystems 7500 Fast Real-Time PCR system (Applied Biosystems, Waltham, MA, USA) following the manufacturers’ instructions. The β-Actin gene was used as the endogenous control. NbMLP28 and β-Actin were amplified using primer pairs MLP28 QF/MLP28 QF and β-Actin QF/β-Actin QR, respectively (Supplementary Table 1). Meanwhile, two PVY primers, PVY-F and PVY-R, were used to detect the changes in virus coat protein expression. The − 2-△△CT method was used to calculate the relative expression level of target gene and three biological replicates were analyzed for each line [41].
Western blotting
For western blotting, protein was isolated from N. benthamiana, and total plant proteins were 1:1 equal volume mixed with 2 × SDS-PAGE buffer. Next, the protein samples were incubated at 95 °C for three min and separated on a 12% SDS-polyacrylamide gel. The separated proteins were then transferred onto nitrocellulose membranes by electroblotting instrument. The PVY CP antibody (SRA20001, Agdia, USA), anti-RFP (ab62341, Abcam, Shanghai) and β-Actin (CW0264M, CWBIO, Beijing) antibody were used for this assay.
Morphological characterization of the transgenic plants
Seeds of the wild-type and NbMLP28::RFP overexpression N. benthamiana lines received in the same batch were surface-sterilized and sown on one-half Murashige & Skoog plates. The plates were stratified at 4 °C for 24 h and let grow vertically at 25 °C with a 16 h/8 h light/dark photoperiod to examine root morphology. Plant root of the plants was measured over a one-week period. These experiments were repeated three times with 100 plants of each of the WT and NbMLP28::RFP lines were used per replicate.
Statistical analysis
Mean values of at least three independent experiments are shown and standard deviations (S.D.) are given. Duncan’s multiple range test analysis of variance (ANOVA), and independent sample t-test were performed in SPSS (v.21, IBM, Armonk, NY, USA). P < 0.05 denotes significant differences between comparisons.
