Enhanced tolerance to Phytophthora root and stem rot by over-expression of the plant antimicrobial peptide CaAMP1 gene in soybean

Background Antimicrobial peptides play important roles in both plant and animal defense systems. Moreover, over-expression of CaAMP1 (Capsicum annuum antimicrobial protein 1), an antimicrobial protein gene isolated from C. annuum leaves infected with Xanthomonas campestris pv. vesicatoria, confers broad-spectrum resistance to hemibiotrophic bacterial and necrotrophic fungal pathogens in Arabidopsis. Phytophthora root and stem rot (PRR), caused by the fungus Phytophthora sojae, is one of the most devastating diseases affecting soybean (Glycine max) production worldwide. Results In this study, CaAMP1 was transformed into soybean by Agrobacterium-mediated genetic transformation. Integration of the foreign gene in the genome of transgenic soybean plants and its expression at the translation level were verified by Southern and western blot analyses, respectively. CaAMP1 over-expression (CaAMP1-OX) lines inoculated with P. sojae race 1 exhibited enhanced and stable PRR tolerance through T2–T4 generations compared with the wild-type Williams 82 plants. Gene expression analyses in the transgenic plants revealed that the expression of salicylic acid-dependent, jasmonic acid-dependent, and plant disease resistance genes (R-genes) were significantly up-regulated after P. sojae inoculation. Conclusions These results indicate that CaAMP1 over-expression can significantly enhance PRR tolerance in soybean by eliciting resistance responses mediated by multiple defense signaling pathways. This provides an alternative approach for developing soybean varieties with improved tolerance against soil-borne pathogenic PRR.

CaAMP1, an AMP gene isolated from pepper (C. annuum) leaves infected with Xanthomonas campestris pv. vesicatoria, has been implicated in broad-spectrum resistance to the hemibiotrophic bacterial pathogen Pseudomonas syringae pv. tomato, biotrophic oomycete Hyaloperonospora parasitica, and fungal necrotrophic pathogens Fusarium oxysporum f. sp. matthiolae and Alternaria brassicicola [8]. Over-expression of CaAMP1 enhances tolerance to hemibiotrophic bacterial and necrotrophic fungal pathogens in Arabidopsis, when compared with the wild-type plants, and induces two salicylic acid (SA) pathway-dependent genes, i.e., PR1 (PATHOGENESIS-RELATED) and PR5 expression [8]. Defensin genes isolated from rice (OsDEF7 and OsDEF8) have been shown to inhibit the phytopathogens X. campestris pv. glycines, X. oryzae pv. oryzicola, and Erwinia carotovora subsp. atroseptica, and weaken the activity of the phytopathogenic fungi Helminthosporium oryzae and F. oxysporum f. sp. cubense [7,9]. Protein extracts with MSI-99, an AMP expressed in chloroplasts of tobacco, could significantly suppress two rice blast isolates, both in vitro and in vivo [10]. Furthermore, leaf extracts from transplastomic tobacco are shown to inhibit the growth of pregerminated spores of three fungal species, Aspergillus flavus, F. moniliforme, and Verticillium dahliae [3], whereas expression of the AMP alfAFP in transgenic greenhouse-grown potato confers tolerance against V. dahliae, an agronomically important fungal pathogen [11]. In addition, AMPs have also been demonstrated to enhance tolerance to fungal diseases in bentgrass and citrus [12,13]. Collectively, these findings evidence that AMPs play key roles in plant defense against fungal pathogens, and that over-expression of these peptides can enhance tolerance against many fungal diseases. Soybean (Glycine max L. Merr.) is an economically important crop worldwide, acting as a rich source of vegetable oil and protein for both humans and livestock. Phytophthora stem and root rot (PRR), caused by the soil-borne hemibiotrophic oomycete Phytophthora sojae, is one of the most devastating fungal diseases in soybean, resulting in an annual yield reduction and economic loss of approximately 10-50% and 1-2 billion dollars, respectively [15][16][17][18]. PRR caused by P. sojae has become a major threat to soybean production in China since it was first reported in 1989 [15,19]. Current measures for controlling PRR in the fields include drainage improvement, crop rotation, and fungicide application. Host-mediated resistance provided by "resistant to P. sojae" (Rps) genes has also been employed to improve PRR tolerance in soybean [16,[20][21][22][23][24][25]. However, Rpsmediated resistance to PRR in soybean may be lost over long periods owing to the high variability of P. sojae, with at least 55 races identified to date [26]. Other alternative control methods include the development of transgenic soybean with enhanced PRR tolerance achieved by the increased levels of pathogenesis-related (PR) proteins, such as Gly m 4 l and ethylene response factor [27], or harpin protein-encoding genes [28,29].
As mentioned previously, AMP over-expression confers broad-spectrum resistance against bacterial and fungal pathogenic infections in plants, suggesting that AMPs may enhance soybean resistance to PRR. Therefore, we synthesized CaAMP1 and introduced it into soybean via Agrobacterium-mediated transformation, and then evaluated the tolerance of CaAMP1-OX soybean to PRR. We found that over-expression of CaAMP1 enhanced soybean tolerance to PRR, and induced the expression of genes involved in SA-and JAdependent pathways and R-gene signaling.

Generation and screening of transgenic plants
The nucleotide sequence of the CaAMP1 (GenBank ID: AAT35532.1) was synthesized and subcloned into a pCambia3300 vector (Fig. 1a). Constitutive expression of CaAMP1 was induced in the soybean plants under the control of CaMV 35S promoter. Constitutive expression was desirable because P. sojae can infect soybean at various developmental stages in much of the growing season.
Transgenic soybean was generated via Agrobacterium-mediated transformation, with the Williams 82 cultivar as the recipient.
A total of 185 phosphinothricin N-acetyltransferase (PAT)-tolerant plants were generated and screened using the LibertyLink strip test, and the positive ones (indicated by two red lines in Fig. 1b) were grown in a greenhouse to produce seeds. Six T 3 transgenic soybean lines were selected for PCR and Southern blot analyses. The PCR confirmed that T 3 transgenic soybean lines contained CaAMP1 and bar genes (Fig. 1c). Southern blot analysis using CaAMP1 as the probe further confirmed that CaAMP1 was integrated into the genome of transgenic soybean, with approximately 1 to 5 copies of insertions (Fig. 1d). The size of all detected bands was greater than the expected fragment size of 1.98 kb, which covered the sequence between the right border and the unique EcoR I site. In contrast, no signal was detected in the wild-type Williams 82 plants. These results indicated stable integration of CaAMP1 in transgenic soybean.
RT-PCR and western blot analyses were further performed to detect CaAMP1 expression in the six T 3 transgenic soybean lines. A 275-bp fragment was   in all six transgenic lines by RT-PCR, which was absent from the wild-type Williams 82 (Fig. 2a). We also detected 20.99 kDa bands in the six transgenic lines, which was absent from the wild-type plants, confirming the expression of CaAMP1 at both transcriptional and translational levels in the transgenic soybean (Fig. 2b). These results indicated that CaAMP1 was successfully transformed into the soybean, and accurately transcribed and translated in the six transgenic lines.

Stable and enhanced tolerance of transgenic soybean to PRR
The PRR tolerance of T 3 transgenic lines and wild-type soybean was evaluated after inoculation of greenhousegrown plants with P. sojae race 1. As shown in Fig. 3, the tolerance of transgenic lines to PRR was markedly enhanced, when compared with that of the wild-type control and Jiunong 21 (JN21) plants which was susceptible to P. sojae (Fig. 3). Typical symptoms of PRR were visible in wild-type Williams 82 and JN21 5 to 10 days after inoculation with P. sojae race 1 mycelia, with some plants succumbing to the progressing pathogenic infection ( Fig. 3a), which was more prominent in JN21. In contrast, most transgenic lines were only slightly affected, as evident from the development of chlorotic leaves (Fig. 3a). Survival rates of transgenic lines (66.17-94.68%) over three generations were higher than those of wild-type Williams 82 (43.67-56.17%) and JN21 (0-8.08%), which was consistent with the results of PRR tolerance (Fig. 3b). Of the six transgenic lines, four (8096, 8101, 8197, and 8253) exhibited a stable enhancement in the tolerance to P. sojae race 1, when compared with the JN21 and wild-type controls (Fig. 3b). In conjunction, these results suggest that CaAMP1-OX transgenic soybean were more tolerant to PRR.
Up-regulation of disease-responsive genes in CaAMP1-OX transgenic soybean CaAMP1 has been shown to induce PRs expression in transgenic Arabidopsis [8]. In the present study, the transcription of 15 defense-related genes involved in SAand JA-dependent pathways, and R-genes was assessed in two transgenic lines (8096 and 8253) after inoculation with P. sojae race 1. The expression levels of SAdependent pathway genes GmPR1, GmPR2, GmPR3, GmPR5, GmPR12, GmPAL, and GmNPRs, were significantly higher in the two transgenic lines than in the wild-type soybean (Fig. 4a-h). Two JA-dependent pathway genes, GmAOS and GmPPO, also exhibited increased expression in these lines ( Fig. 4i and j). Moreover, the expression of both R-genes, i.e., GmSGT1 and GmRAR1 (Fig. 4k and l), involved in plant resistance to disease, was significantly up-regulated in comparison to the wild-type control. These results indicated that over-expression of the CaAMP1 gene in soybean could elicit multiple resistance responses mediated by different signaling pathways, enhancing plant tolerance against P. sojae infection.
No obvious differences in agronomic traits between CaAMP1-OX and wild-type soybean To evaluate the effect of over-expression of CaAMP1 in soybean, agronomic traits, including maturity period, leaf shape, flower color, hilum color, plant height, branch number, node number, podding height, and 100-seed weight, of field-grown T 3 transgenic lines were analyzed. No differences could be detected in these traits between the transgenic lines and wild-type Williams 82 (Table 1). These results indicated that CaAMP1 over-expression conferred transgenic soybean with enhanced and stable tolerance to PRR without any negative effects on its agronomic traits.
Data on the agronomic traits of wild-type Williams 82 and the six transgenic lines (8096, 8101, 8111, 8130, 8197, 8253) were collected from the experimental station at Gongzhuling, Jilin Province, China. Twenty plants of each line were randomly selected for the measurements. Differences were considered significant at P < 0.05.

Discussion
PRR is one of the most devastating diseases in soybean, resulting in annual losses of 1-2 billion dollars worldwide [15,16]. Previous studies have confirmed that exogenous resistant genes can enhance PRR tolerance in soybean [28,29]. In the present, an AMP gene, CaAMP1, which has previously been demonstrated to confer broad-spectrum resistance against hemibiotrophic bacterial and necrotrophic fungal pathogens in transgenic Arabidopsis [8], is transformed into the soybean genome. We obtain six transgenic lines with enhanced tolerance against P. sojae race 1 over three generations, compared with the wild-type plants. Furthermore, we observe an up-regulation of several defense-related genes in these transgenic lines. These observations indicate that CaAMP1 plays a functional role in stimulating defense-related genes involved in SA-dependent, JAdependent, and R-defense signaling in response to pathogenic infection.
SA-and JA-dependent signaling pathways are essential for defense against pathogens. SA is crucial to immune responses against biotrophic and hemibiotrophic pathogens [30,31], and also involves in cross talks between ethylene and methyl jasmonate signaling pathways [32,33]. SA-dependent defense pathways can be induced when pathogen infects the plant and finally activate on pathogenic infection in plants, subsequently activating the PRs [34]. Over-expression of CaAMP1 protein in Arabidopsis triggers rapid expression of AtPR1 and AtPR5 after infection with the virulent strain Pst DC3000, enhancing plant resistance to this disease [8]. In addition to PR genes, three other SA-dependent pathways genes, i.e., PAL, NPR1-1, and NPR1-2 are also upregulated in the CaAMP1 transgenic lines in the present study. PAL is required in SA bio-synthesis pathways [35][36][37], and NPRs are indispensable for cross talks between SA and JA/ethylene responses [38]. Overexpression of these two genes can enhance disease tolerance in soybean and Arabidopsis [39,40]. Furthermore, the activation of SA-dependent PR genes is observed to be more rapid and intense in NPR1-OX transgenic plants than in their wild-type counterparts [40]. These genes can also be up-regulated in transgenic soybean by exogenous oxalate oxidase, hrpZm, and hrf2, after infection with P. sojae race 1 [28,29,37]. JA-regulated defense is an important component of plant resistance to necrotrophic fungi [32,41,42]. AOS and PPO, involved in JA-dependent signaling pathway, are induced in CaAMP1-OX soybean lines. This is not consistent with the observations made in Arabidopsis [8], which may be attributed to the different genes selected in the present study. SGT1 and RAR1 are important genes in plant resistance signaling pathways [43][44][45], which are also up-regulated in CaAMP1-OX transgenic soybean.
Collectively, these results suggest that CaAMP1, as an AMP, can enhance PRR tolerance in transgenic soybean by triggering the production of SA-and JA-dependent defense signaling molecules and R-genes. These results confirm that rapid induction of SA and JA signaling pathways is associated with early recognition of the   pathogen and resistance in soybean [46]. Furthermore, we do not observe differences in the agronomic traits between CaAMP1-OX lines and their wild-type counterparts.

Conclusions
The results of the present study indicated that overexpression of CaAMP1 in soybean can significantly enhance PRR tolerance by inducing the expression of resistant genes involved in multiple defense signaling pathways. This may provide an alternative approach for developing soybean varieties with improved tolerance against soil-borne pathogenic PRR.

CaAMP1 synthesis and vector construction
The nucleotide sequence of C. annuum CaAMP1 (Gen-Bank ID: AAT35532.1) was synthesized with added Xba I and Sac I recognition sites at the 5′ and 3′ ends, respectively (Sangon Biotech, Shanghai, China). The modified CaAMP1 gene was inserted into a pCambia3300 vector containing a modified CaMV 35S promoter [47] (GenBank: GI3319906) to facilitate its constitutive expression in soybean. The gene sequence was amplified using the CaAMP1-F/R primer pair, with a final primer concentration of 0.4 μM, under the following conditions: 94°C for 5 min; followed by 35 cycles of 94°C for 30 s, 59°C for 30 s, and 72°C for 30 s; and final extension at 72°C for 7 min. All primers used in this study were listed in Table S1. The purified fragment was then subcloned into a pCambia3300 plasmid containing a phosphinothricin acetyl transferase (bar) resistance gene, encoding PAT, as a plant selection marker driven by a modified CaMV 35S promoter [47] (GenBank: GI3319906). The constructed pCambia3300-CaMV 35S-CaAMP1 plasmid was subsequently transformed into competent A. tumefaciens strain EHA101 cells, by the freeze-thaw method [48,49].  (Table S1) until homozygous transgenic plants were obtained. DNA was extracted from the leaves of transgenic and wild-type soybean, using a simple homogenization and ethanol precipitation method, for PCR analysis [52]. PCR was performed with a final primer concentration of 0.2 μM, with the following conditions: 94°C for 5 min; followed by 35 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s; and final extension at 72°C for 7 min.

Regeneration and screening of transgenic plants
To confirm the integration of T-DNA in transgenic soybean, T 2 transgenic plants were selected for genomic DNA extraction, using a modified high salt cetyltrimethyl ammonium bromide method [53]. DIG High Prime DNA Labeling and Detection Starter Kit I (No. 11745832910; Roche Applied Science, Indianapolis, IN, USA) was used for Southern blot analysis, according to the manufacturer's instructions. Approximately 30 μg of the genomic DNA from transgenic soybean and control plants was digested completely with EcoR I (New England Biolabs Inc., Beverly, Massachusetts). The digested DNA was then transferred onto positively charged nylon membranes (GE Amersham, RPN303B, USA). Hybridization was carried out at 42°C for 12-16 h, using CaAMP1 labeled with digoxigenin-(DIG)11-dUTP as a probe. The washing conditions and signal detection were as described in Yang et al. (2018) [50].

Evaluation of PRR tolerance under greenhouse conditions
To evaluate the tolerance of transgenic soybean against P. sojae race 1, the T 2 − T 4 generations of transgenic lines 8096, 8101, 8111, 8130, 8197, and 8253 were infected with P. sojae race 1, following the method described by Schmitthenner et al. (1994) [55]. Isolation and cultivation of the inoculum were performed as described by Akamatsu et al. (2010) and Du et al. (2018) [28,56]. Transgenic soybean, wild-type Williams 82, and the PRR-susceptible cultivar Jiunong 21 (ID:ZDD22796), which were provided by the Soybean Research Institute of Jilin Academy of Agricultural Sciences, were grown in a greenhouse, and the hypocotyls of 15-day-old seedlings were inoculated with macerated mycelia of P. sojae race 1. The plants were then maintained in a humid environment for 15-24 h, before being transferred to the greenhouse for symptom development, at 25°C under an 18-h light/6-h dark photoperiod [28]. After 5 to 10 days of inoculation, plant infection data were collected and survival rates were calculated [57]. All experiments were performed with three replicates of 20 inoculated plants each replicate.
Differences in the survival rates of the control and transgenic lines were quantitatively assessed by t-test at a significance level of P = 0.05 or 0.01, using Microsoft Analysis Tool.
To improve the accuracy of the data, three biological and three technical replicates were performed for each experiment.

Agronomic traits of transgenic lines
Nine agronomic traits of T 3 transgenic lines and wildtype soybean were assessed, including maturity period, leaf shape, plant height, flower color, hilum color, branch number, node number, podding height, and 100seed weight, and t-test was used for quantitative analysis.
Additional file 1. Supplementary Table 1 Abbreviations