Morus multicaulis (Husang 32) was incubated in a growth chamber at 26°C, humidity 50–60% and 12 h of light. Arabidopsis thaliana (Col-0) plants were grown at 22°C, humidity 50–60% and 12 h of light.

RNA was isolated from leaves of M. multicaulis using TRIzol^® reagent following the manufacturer’s recommendations (Invitrogen) and digested with DNase I. cDNA was synthesized using 2 μg of total RNA with 100 units of reverse transcriptase M-MLV (Promega) in 20 μL reactions. The specific oligonucleotide primers (MLX56-5′: 5′-GCATGAAGTTTAGAACTCTTCT-3′; MLX56-3′: 5′-TTACATTCGAGCAACTTCCG-3′) were designed based on our available mulberry transcriptome data for PCR amplifications, and the DNA fragments obtained from RT-PCR were subcloned individually into the pMD18-T vector (Invitrogen) resulting in pMD18-MLX56. After transformation, positive clones were selected and further sequenced. The deduced amino acid sequences of MLX56 genes were aligned using DNAMAN multiple alignments program. Structural prediction was performed with SWISS-MODEL tools¹.

To obtain the promoter sequence of MLX56 from Husang 32, chromosome walking was performed using the TAIL-PCR method. Three specific primers (SP1: 5′-TTGGCAACCTTGATCAACATCACA-3′, SP2: 5′-ACGATGGACTCCAGTCGGCATAAGGCACCTCCTACA-3′ and SP3: 5′-TGTTGCTCACTACAATTTCTAGCAGAA-3′) were designed using the MLX56 cDNA sequence. Four arbitrary primers were used (LAD-1:5′-ACGATGGACTCCGVNVNNNGGAA-3′; LAD-2: 5′-ACGATGGACTCCAGAGCGBNBNNNGGTT-3′; LAD-3: 5′-ACGATGGACTCCAGAGCGBDNBNNNCGGT-3′; LAD-4: 5′-ACGATGGACTCCAGAGCGHNVNNNCCAC-3′) and the AC1 (5′-ACGATGGACTCCAGAG-3′) primer complementary to an adaptor sequence within the LAD primers was designed according to the previous study (Liu and Chen, 2007). Mulberry genomic DNA was isolated using the cetyltrimethyl-ammonium bromide method as previously described (Sato et al., 1996) and subjected to preamplification using the primers LAD and SP1. The amplification product was diluted and used as the template in the primary TAIL-PCR using the primer pairs AC1 and SP2. By using the same method, the primary TAIL-PCR product was used as a template in the secondary TAIL-PCR using the primers AC1 and SP3. The secondary TAIL-PCR amplified products were analyzed on 1.0% agarose gels, and the bands of interest were isolated and purified using a universal genomic DNA extraction kit (TaKaRa) and ligated to pMD18-T vector, then transformed into Escherichia coli for sequencing. The potential cis-regulatory elements within the promoter were analyzed using the PlantCARE program online².

The promoter was cloned into the vector pBI121 to replace the cauliflower mosaic virus (CaMV) 35S promoter and fused to the GUS (β-glucuronidase) reporter gene to create the promoter expression vector pHMLX56::GUS, and the derived construct vector was introduced into Agrobacterium tumefaciens strain GV3101. For Arabidopsis transformation, the floral dipping method (Clough and Bent, 1998) was employed. Histochemical staining for GUS activity was performed as described by Jefferson (1987).

Two-week-old transgenic and wild-type Arabidopsis seedlings were grown in the growth chamber under conditions specified above. The abscisic acid (ABA), gibberellin (GA) and salicylic acid (SA) treatments were achieved by spraying the rosette leaves with 100 μM ABA, 200 μM GA, or 5 mM SA solution. Control plants were sprayed with water. To test the effects of pathogen stimuli on the activity of the promoter, Arabidopsis seedlings were inoculated with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) by spraying Pst DC3000 bacterial suspensions (10⁵ CFU mL^-1) onto the rosette leaves. Water was used as control. All samples from the above treatments were harvested 12 h post-treatment. For wounding treatment, the leaves were squeezed with a tweezer, and the wounded seedlings were harvested 4 h later. For dark and light treatments, seedlings were incubated continuously in the dark or light for 24 h. Each treatment was replicated three times with 10 seedlings per replicate. All the samples described above were immediately frozen in liquid nitrogen after harvest and stored at -80°C for GUS fluorometric assays.

To assay GUS activity, Arabidopsis leaves were harvested and stored at -80°C before use. Frozen leaves were ground in extraction buffer (50 mM pH 7 sodium phosphate, 10 mM EDTA, 0.1 Sarkosyl, 0.1 M Triton X-100, and 10 mM β-mercaptoethanol). The homogenate was centrifuged at 12,000 × g for 15 min (4°C), then the supernatant was used for GUS activity assays. GUS activity was assayed by using 10 μL extract and 4-methyl-umbelliferyl-glucuronide (Sigma) as a substrate. The protein concentration of the extracts was determined utilizing bovine serum albumin as a standard protein according to the assay described by Bradford (1976). Fluorescence was measured in a fluorescence spectrophotometer (Hitachi, Tokyo, Japan). The excitation wavelength was 365 nm and the emission wavelength 455 nm. Each assay was repeated three times. The data presented were collected from at least three independent experiments.

Total RNAs were extracted from leaves, stems, roots, flowers and fruits of M. multicaulis (Husang 32) plants, and cDNA was synthesized as described above. Real-time PCR was performed using the SYBR Premix Ex Taq^TM kit (TaKaRa) according to the manufacturer’s protocol on the Rotor-Gene 3000A system. The EF1-α gene was amplified as a reference gene for mRNA normalization. The MLX56 gene was amplified using primer pair F (5′-TGTAATCCAGGAAGGTGTTGTAG-3′) and R(5′-GAGAAGTCCAACATTGGTATTG-3′), and the EF1-α gene was amplified using primer pair F (5′-ATGGTGAAGATGATTCCCACTAAGC-3′) and R (5′-AAAAGCCAGTCACTTCCCTCCCT-3′). Comparative cycle threshold (Ct) method (Livak and Schmittgen, 2001) was used to evaluate the relative gene expression level. All samples were assayed in triplicate.

The coding region of the MLX56 gene was amplified from pMD18-MLX56 plasmid DNA with Xba I sense primer (5′-TCTAGAATGAAGTTTAGAACTCTT-3′) and Sac I antisense primer (5′-GAGCTCTTACATTCGAGCAACT-3′). The PCR-amplified fragment was cloned into pMD18-T plasmid, which was then digested with Xba I and Sac I. The products were analyzed by 1% agarose gel electrophoresis, and a DNA fragment of approximately 1200 bp was recovered and subcloned into binary plasmid vector pBI121 (digested with Xba I and Sac I) under the control of the 35S promoter. Then, the vector was introduced into A. tumefaciens strain GV3101, and wild-type Arabidopsis plants were transformed with the floral dip method. After transformation, the sterilized T1 seeds were plated on kanamycin selection plates (MS media supplemented with 50 μg mL^-1 kanamycin) to select transformed plants.

For northern blotting, total RNA was extracted, separated and then blotted onto a nylon Hybond N membrane. The blots were hybridized with digoxigenin-labeled RNA probes prepared using the PCR DIG Probe Synthesis Kit (Roche). Prehybridization, hybridization, membrane washing, and detection were performed according to the method described by Umezawa et al. (2006). For western blotting, protein was prepared and mixed with 5 × SDS-PAGE sample buffer. Then, the samples were heated at 95°C for 3 min and separated by SDS-PAGE on 12% SDS-polyacrylamide gels. After electrophoresis, the separated proteins were electroblotted onto nitrocellulose membranes. Western blot analysis was performed using the anti-MLX56 protein polyclonal antibody according to a previously described method (Poppenberger et al., 2011).

Surface-sterilized Arabidopsis seeds from transgenic Arabidopsis lines expressing HMLX56 gene and wild-type were sown on MS media and kept in darkness for 2 days at 4°C to synchronize germination. Then the seeds were germinated on MS medium and grown vertically in a growth chamber at 22°C on a 16/8 h light/dark cycle for root morphology examination. At the same time, the seeds were planted in square plastic pots filled with mixed soil (vermiculite: humus = 1:1) and cultured under the same condition. Height, number of rosette leaves and shoots, and days to flowering were measured over a 6-week period. These experiments were carried out with three replications and six plants per replication.

Plutella xylostella tests The eggs of P. xylostella were hatched in an incubator at 26°C, and caterpillars of approximately 1 mg in size were placed on the leaves of 4-week-old Arabidopsis plants with two individuals per plant. The weights of caterpillars were determined 6 days later. This experiment was conducted with three replicates and 10 seedlings per replicate.

Aphid tests Stock colonies of Myzus persicae were reared on Chinese cabbage (Brassica rapa, ssp. chinensis) under standard conditions in a growth incubator at 25°C, 60% relative humidity and 16 h photoperiod. Synchronized 1-day-old nymphs were used to infest 4-week-old Arabidopsis plants with two nymphs per plant. The average weight of aphids on plants was measured at 5 days after introduction, and the total number of nymphs was calculated every 4 days for 16 days after introduction. Three replications of this experiment were carried out with 10 seedlings per replicate.

Four-week-old transgenic Arabidopsis and wild-type plants were used for disease resistance analysis. Leaves of each plant were detached with a sharp blade and challenged with Botrytis cinerea by applying 2 mm diameter plugs of PDA media containing actively growing mycelia of B. cinerea. The leaves were then placed on wet filter paper in a covered culture dish to maintain high humidity, and incubated at 22°C. Disease incidence and lesion sizes, presented as the diameter of the lesion (mm), were surveyed. Inoculation with Pst DC3000 was conducted by infiltrating with 50 μL bacterial suspensions (10⁵ CFU mL^-1) using 1-mL syringes without needles, and the disease symptoms were recorded using a camera. Chlorophyll abundance was measured to assay chlorosis development in the inoculated leaves. Leaf disks from three separate leaves were frozen in liquid nitrogen and then homogenized in 80% (v/v) acetone. The homogenates were centrifuged at 500 × g for 3 min at 4°C, and the supernatant was used for chlorophyll assays. The amount of chlorophyll was determined on a scanning spectrophotometer as previously described (Ritchie, 2006). Each treatment was replicated three times with 10 seedlings per replicate.

Data are reported as means ± SD of at least three independent experiments. Statistical significance was subjected to Duncan’s multiple range test with analysis of variance (ANOVA) using Statistical Analysis System software (v. 9.3 for Window; SAS Institute, Cary, NC, United States, 2010).

The MLX56 gene was isolated from M. multicaulis by PCR and designed as HMLX56 (Bank Accession No. JX432966). The open reading frame (ORF) encoded a protein of 400 AA which shows 91 and 93% amino acid sequence identity with those of M. notabilis (Chuansang) and M. alba (Shin-Ichinose), respectively (Figure 1). The signal peptide (amino acid residues 1–21) predicted by SIGNALP V3.0 in the N-terminal region of HMLX56 protein was identical to those of other MLX56 proteins (Figure 1). When the signal peptide was removed, the mature MLX56 proteins of M. multicaulis, M. notabilis, and M. alba had similar structures comprising three domains. The first part, consisting of two hevein-like chitin-binding domains, was well conserved between different Morus species, and this domain had eight conserved cysteine residues which were essential for keeping the functional structure by four intrachain disulphide bridges. Though the numbers of Ser[Pro]_n repeats in the middle of the two chitin-binding regions were different between HMLX56 and the other two MLX56 proteins, the extensin domain (motif) was well conserved among the three MLX56 proteins. In addition, the third part, consisting of a C-terminal chitinase-like domain, was also well conserved among the three proteins (Figure 1). Molecular modeling results indicated that the spatial architecture of the HMLX56 protein is very similar to those of the MLX56 proteins from Chuansang and Shin-Ichinose (Figure 2). These data revealed that the MLX56 proteins are conserved among Morus species, and they may have similar functions.

To obtain better insight into the biological function of the HMLX56 gene, we first investigated its expression levels in different organs of Husang plants. The RT-PCR analysis indicated that the HMLX56 gene was constitutively expressed in mulberry organs, but its expression level varied considerably across organs (Figure 3A). The HMLX56 gene was highly expressed in leaves and bark, but it was expressed at a low level in flowers and fruits and at the limits of detection in the roots. To explore the precise expression patterns of HMLX56 at the tissue level, the putative promoter, 1848 bp DNA upstream of the HMLX56 coding region sequence, was cloned (designated pHMLX56), and a plant expression vector containing pHMLX56 promoter fused to GUS gene was constructed and introduced into Arabidopsis plants. These transgenic plants were subjected to histological GUS analysis to investigate the expression location of HMLX56 gene. Strong GUS signals were detected around the vascular cylinder of leaves indicating that the HMLX56 gene was a tissue-specific gene and might be expressed specifically around the vascular cylinders (Figure 3B). Cis-acting regulatory elements analysis of the sequence of pHMLX56 showed that it contains some cis-acting elements involved in the light, ABA, wounding and pathogen responses (Table 1). To investigate whether these environmental factors were involved in the regulation of pHMLX56 activity, the regulatory patterns of pHMLX56 under treatment with ABA, SA, GA, Pst DC3000, wounding, light and dark were analyzed. Using fluorometry, an obvious induction of GUS activity was observed in pHMLX56 plants upon treatment by SA, ABA, Pst DC3000 and wounding, and lower GUS activity was observed in the pHMLX56 plants upon treatment by light. No change in GUS activity was observed upon dark treatment. In contrast, when exposed to GA, GUS activity was significantly decreased (Figures 3C,D). These results indicate that HMLX56 promoter activity can be regulated by ABA, SA, GA, Pst DC3000, wounding, and light, but with different activity levels in response to different environmental factors.

Transgenic Arabidopsis plants constitutively expressing the HMLX56 gene were generated by transforming wild-type Arabidopsis plants with constructs containing the HMLX56 ORF sequence under the regulation of the 35S promoter. The HMLX56 gene was successfully integrated into the Arabidopsis genome (Figure 4A) and expressed at detectable mRNA and protein levels in the transgenic Arabidopsis plants (Figures 4B,C). All the HMLX56 over-expression lines did not show significantly different root morphology, number of rosette leaves and shoots, and flowering time compared with the wild-type plants (Figures 4D–F). This indicated that ectopic expression of HMLX56 in Arabidopsis plants has no effect on plant growth and development, and the HMLX56 gene may not be involved in the process of plant development.

To evaluate the insecticidal activity of HMLX56, 4-week-old wild-type and transgenic (ectopically expressing the HMLX56 gene) Arabidopsis plants were challenged with P. xylostella caterpillars of approximately equal developmental stages and weight, and the weight of caterpillar was determined 6 days later. The results showed that the caterpillars feeding on wild-type Arabidopsis exhibited significantly increased mean weight in comparison to those feeding on transgenic plants (Figure 5A). Therefore, the HMLX56 protein exhibits significant growth-inhibitory activity against P. xylostella caterpillars. To further investigate the role of HMLX56 protein in insect resistance, we studied the resistance of the HMLX56-overexpressing transgenic Arabidopsis to green peach aphid. Two first-instar aphids were transferred to each of the wild-type and transgenic plants grown in the same pot. The average weight of aphids on the plants was measured 5 days later, and the results showed that the average weight of aphids feeding on transgenic plants was significantly lower than that of aphids feeding on the wild-type plants (Figure 5B). In addition, the number of aphids was also counted 16 days after introduction, and the population of aphids feeding on the transgenic plants was significantly smaller than that of aphids feeding on the wild-type plants (Figure 5C). These data suggested that the expression of HMLX56 gene in Arabidopsis enhances the resistance of transgenic plants to aphids. Therefore, HMLX56 exhibits significant growth-inhibitory activity against both P. xylostella caterpillars and aphids.

To examine the role of the HMLX56 gene in plant defense response to pathogens, transgenic Arabidopsis plants overexpressing the HMLX56 gene were inoculated with B. cinerea and Pst DC3000. When the detached leaves from 4-week-old Arabidopsis plants were inoculated with B. cinerea, disease development was analyzed 4 days after inoculation (DAI). The results showed that leaves of wild type plants inoculated yielded expanding disease yellow lesions around the inoculated points. However, the disease lesions around the inoculated points in the inoculated leaves of transgenic plants were smaller compared with those in the wild type plant leaves inoculated (Figures 6A,B). To determine whether the HMLX56 gene is involved in plant response to bacterial pathogen, the plants were inoculated with Pst DC3000. Three days after inoculation, the chloroses surrounding inoculation points in the leaves of wild-type plants were larger and more severe than those in the leaves of transgenic plants (Figures 6C,D), suggesting that the HMLX56 gene conferred resistance to Pst DC3000 in Arabidopsis. To further confirm this, the bacterial growth in the inoculated leaves was determined, and the results showed that the growth of Pst DC3000 strain was extremely limited in the leaves of plants overexpressing HMLX56 (Figure 6E). This was in accordance with the milder symptom development in the plants overexpressing HMLX56. Therefore, ectopically expressing the HMLX56 gene in Arabidopsis enhanced plant resistance to B. cinerea and Pst DC3000, and the HMLX56 gene may have roles in the defense response to fungal and bacterial pathogens.