Salt stress-induced polyamine biosynthesis contributes to blast resistance in rice

Abstract

Rice blast, caused by the fungal pathogen Magnaporthe oryzae, is a destructive disease that affects rice (Oryzae sativa L.) on a global scale. Polyamines (PAs) play diverse roles in plant growth and development and responses to biotic and abiotic stimuli. Putrescine (PUT), spermidine (SPD), and spermine (SPM) are the primary forms of polyamines (PAs). In this study, we observed that the accumulation of apoplastic PAs significantly increased in rice plants after treatment with salt or M. oryzae. The salt-treated plants exhibited enhanced resistance to rice blast disease. RNA sequencing data indicate that S-adenosylmethionine decarboxylase (SAMDC), a key enzyme involved in the synthesis of polyamines, plays a significant role in enhancing plant resistance. Overexpression of rice SAMDC (OsSAMDC) led to a significant decrease of pathogen infection in the transgenic rice plants. Additionally, OsSAMDC overexpression plants accumulated polyamines in the cytosol and apoplast, particularly SPD and SPM. Conversely, the disease resistance and accumulation of PAs were reduced in OsSAMDC-silenced plants. Exogenous application of PAs inhibited the mycelium growth, spore germination, germ tube elongation, and appressorium formation in M. oryzae. These results demonstrated that OsSAMDC-mediated polyamine biosynthesis, especially SPD and SPM, plays an essential role in rice plants to resist biotic and abiotic stresses.

Background

The cultivation of rice (Oryza sativa L.) is constrained by biotic and abiotic stresses, such as salinity, drought, pathogens (bacteria, fungal, and viral), as well as insect herbivory. Roughly, there are 3.6 billion of the 5.2 billion hectares of dry land utilized for agriculture worldwide that are affected by erosion, soil degradation, and salinization (Riadh et al. 2010). Among all the abiotic stresses, about 10% of the land surface (950 Mha) and 50% of all irrigated land (230 Mha) are damaged by salt (Qadir et al. 2008). In addition to the abiotic stresses, rice yield is constantly threatened by many diseases. Of all the diseases in rice, blast disease caused by the fungal pathogen Magnaporthe oryzae results in the most severe yield loss worldwide. Improvement of rice yield under these challenging environments is a goal for breeders and farmers.

Polyamines (PAs) are organic molecules with several amino groups that are positively charged at physiological pH. PAs interact with anionic biological macromolecules, including nucleic acids and phospholipids (Igarashi and Kashiwagi 2010). Therefore, these entities participate in a multitude of cellular functions by affecting gene expression, promoting cell growth, influencing cell communication, and stabilizing cell membranes (Thomas and Thomas 2001; Seiler and Raul 2005; Igarashi and Kashiwagi 2010). Although the biological significance of PAs is widely recognized, understanding the physiological importance of changes in PA metabolism caused by pathogen infection under saline environment is a challenging endeavor. In plants, Putrescine (PUT), Spermidine (SPD), and Spermine (SPM) are the three major types of PAs, and Cadaverine (CAD) is another type of polyamine found in legume plants (Kumar et al. 1997; Walden et al. 1997). Polyamine biosynthesis pathway in Arabidopsis has been well characterized (Alcazar et al. 2006), which is initiated by the sequential reaction of arginine decarboxylase (ADC; EC4.1.1.19), agmatine iminohydrolase (AIH; EC3.4.3.12), and N-carbamoyl putrescine amidohydrolase (CPA; EC3.5.1.53). Polyamine biosynthesis begins with PUT synthesis from amino acid arginine. SPD and SPM are generated when aminopropyl groups are added sequentially to PUT and SPD, respectively, by the enzymes SPD synthase (SPDS; EC2.5.1.16) and SPM synthase (SPMS; EC2.5.1.22). Decarboxylated S-adenosylmethionine (dcSAM), which is produced through the decarboxylation of S-adenosylmethionine (SAM) in a reaction catalyzed by S-adenosylmethionine decarboxylase (SAMDC; EC4.1.1.50), serves as a donor molecule for aminopropyl groups for both enzymes. The accumulation of polyamines in plant tissues after infection with pathogens has been documented (Wimalasekera et al. 2011). For example, when tobacco plants were infected with the tobacco mosaic virus (TMV), the activity of ornithine decarboxylase (ODC) and the amount of free spermine were increased significantly in plants (Negrel et al. 1984; Yamakawa et al. 1998). In addition, when tobacco plants were infected with TMV, the activity of diamine oxidase (DAO) and the expression of polyamine biosynthesis genes were upregulated, which subsequently triggered a hypersensitive response (Marini et al. 2001). In Arabidopsis, spermine effectively inhibits the replication of the cauliflower mosaic virus (CaMV) by triggering a signaling cascade that ultimately activates defense systems (Mitsuya et al. 2009). The accumulation of PA has been documented in tobacco plants inoculated with Pseudomonas viridiflava, and Arabidopsis plants inoculated with Pseudomonas syringae (Angelini et al. 2010). Additionally, PA accumulation has been observed in tobacco infected with Pseudomonas cichorii (Yoda et al. 2009). Overexpression of Arabidopsis SAMDC1 in plants has been observed to elevate spermine content and to increase disease resistance to P. syringae in plants (Marco et al. 2014). Furthermore, fungal infection has an impact on the polyamine metabolism in plants. An increase in the production and breakdown of PA compounds have been seen in barley plants during the hypersensitive reaction caused by the powdery mildew fungus Blumeria graminis f. sp. hordei (Cowley and Walters 2002). Barley plants infected with powdery mildew showed increased activities of polyamine biosynthetic enzymes, leading to higher levels of PUT, SPD, and SPM (Walters et al. 2001). The expression of the polyamine biosynthetic gene ACL5 was upregulated in cotton by the infection of vascular wilt fungal pathogen Verticillium dahliae (Mo et al. 2015).

SAMDC is a key enzyme in PA biosynthesis (Wimalasekera et al. 2011), playing a crucial role in catalyzing the decarboxylation of S-adenosylmethionine (SAM) into decarboxylated SAM (dSAM). This dSAM serves as the aminopropyl donor for the biosynthesis of SPD and SPM (Kakkar and Sawhney 2002; Bitrian et al. 2012). SAMDC overexpression leads to increased levels of SPD and/or SPM in plants, which in turn enhances the plant's tolerance to salinity (Roy and Wu 2002; Hao et al. 2005), drought (Waie and Rajam 2003), cold (Wi et al. 2006), and heat (Cheng et al. 2009). Conversely, when the activity of SAMDC was repressed, the levels of SPD and SPM were reduced (Chen et al. 2014), and shorter plant length, decreased and delayed seed germination, and an impaired ability to tolerate various abiotic stresses such as drought, salinity, and cold were observed (Moschou et al. 2008). The increased expression of SAMDC might enhance the activities of antioxidant protecting enzymes, thereby safeguarding plant cells against oxidative harm by eliminating reactive oxygen species (ROS) (Meng et al. 2021; Zhu et al. 2023). SAMDC-overexpression plants showed enhanced enzyme activity of SAMDC and a notable building up of SPD and SPM in rice leaves (Thu-Hang et al. 2002). Consistent with the hypothesis that in overexpressed plants, SPM may play a crucial role in defense signaling, and exhibit greater resistance to Pseudomonas viridiflava when compared to the wild type (WT). Conversely, SPM deficient mutants exhibit an enhanced susceptibility to pathogen infection (Gonzalez et al. 2011). A comparison of the transcriptomes for SPM-accumulating plants and SPM-deficient mutants showed that many genes only expressed in the SPM-accumulating plants and participate in pathogen perception and defense responses, including several families of disease resistance genes, transcription factors, kinases, and nucleotide and DNA/RNA binding proteins (Gonzalez et al. 2011). Consistently, majority of the up-regulated genes also exhibit increased expression in SPM-accumulating lines with overexpression of the SAMDC1 gene (Marco et al. 2011).

OsSAMDC gene has been well investigated for its role in plant growth, development, and abiotic stress tolerance; however, the relationship between SAMDC and biotic stresses is relatively unknown. In this study, we investigated the effects of polyamines on rice salt tolerance and disease resistance. We first validated that salt and M. oryzae-driven stresses were able to induce OsSAMDC expression in rice. We then demonstrated the apparent localization of OsSAMDC to the nucleus, cytoplasm, and plasma membrane. More significantly, we found that transgenic rice plants overexpressing OsSAMDC displayed enhanced resistance to M. oryzae and higher accumulation of apoplastic polyamines. Consistently, SPD and SPM were found to be accumulated upon both M. oryzae infection and salt stress. Our results suggest that OsSAMDC plays a positive role in blast stress resistance in rice.

Results

Salt-treated rice plants showed enhanced resistance to M. oryzae

To understand whether the salt treatment affects the response to biotic stress, we treated the rice plants with different concentrations of salt (50 mM and 100 mM NaCl). Then, the plants were sprayed with M. oryzae (Guy11) conidial suspension. At 5 days post inoculation (dpi), salt-treated plants exhibited enhanced resistance to blast fungus than the WT (Fig. 1a). Moreover, significantly fewer lesions and lower fungal biomass were observed on the leaves of salt-treated plants in comparison with WT (Fig. 1b, c). Compared to Guy11-treated plants, the lesion number decreased by 51.43% and 63.08% at 50 mM and 100 mM salt-treated rice seedlings, respectively. Despite demonstrating significantly enhanced disease resistance at both salt concentrations, the rice seedlings were weakened when treated with a high concentration of salt (100 mM). This result suggests that salt treatments induce stress tolerance or resistance genes expression to regulate the activation of defense responses in plants.

Fig. 1

Salt treatments enhance the blast disease resistance of rice leaves. a Phenotype of rice plants (Nipponbare) under salt and Guy11 treatments. Rice seedlings were treated with 50 mM and 100 mM NaCl for 24 h and then spray-inoculated with conidial suspensions (1 × 10⁵ conidia/mL in 0.02% Tween 20) of M. oryzae. Images were taken at 5 dpi. b, c Estimation of lesion number and fungal biomass in a. The fungal biomass was determined by qPCR of M. oryzae Pot2 gene against the rice OsACTIN gene. Values are mean ± SE. Significant differences from the WT were analyzed using Student's t-test (** p < 0.01)

Expression analysis of polyamine biosynthesis genes in rice under stressed conditions

In order to examine the roles of disease resistance in plants following salt and M. oryzae treatments, we performed transcriptome analysis (RNA-seq) on 50 mM NaCl-treated plants, M. oryzae-infected plants at 24 hpi, and M. oryzae-infected plants which were pretreated with 50 mM NaCl. Based on the criteria |fold change|> 2 and p-adjust < 0.05, we identified 2020, 141, and 1220 differentially expressed genes (DEGs) in leaves after NaCl treatment, pathogen inoculation, and co-treatment when compared with the untreated plants, respectively. Of these, 18 DEGs responded to all three treatments (Fig. 2a). Although polyamines are widely distributed in plants and animals, their role in abiotic and biotic stresses is not well known. We noticed that some genes involved in polyamine biosynthesis were upregulated in response to salt or salt and M. oryzae co-treatment, as revealed by the transcriptome data (Fig. 2b). Among these DEGs, OsSAMDC is one of the polyamine biosynthesis enzymes that was reported to be involved in the response to biotic and abiotic stresses (Islam et al. 2020). Next, we examined the apoplastic polyamine contents of the leaves treated with salt and M. oryzae. The result showed that the salt-treated plants significantly accumulated the highest levels of all three polyamines. Similarly, Guy11-treated plants also have a higher accumulation of polyamines, especially SPD and SPM content, compared to the control. Interestingly, we found that the infected rice plants that were pretreated with salt (Salt 50 mM and Guy11) were observed with elevated PUT, SPD, and SPM contents when compared to the control and Guy11 plants separately (Fig. 2c). These results suggest that salt-stressed plants induce polyamine biosynthesis-related gene expression and the accumulation of higher levels of polyamines as stress tolerance or resistance.

Fig. 2

Transcriptome analysis of the differentially expressed genes response to salt and M. oryzae. a Venn diagram showing the unique number of differentially expressed genes (DEGs) in WT under M. oryzae inoculation and salt treatment. Rice plants were infected by M. oryzae for 24 h and treated with 50 mM NaCl, respectively, and then the transcriptome was analyzed. Differentially expressed genes were detected by DESeq2. DEGs were defined by a cut-off of |logFC|> 1, p-adjust < 0.05 as criteria. b Heatmap analysis of polyamine biosynthesis genes in rice under salt, M. oryzae infection, and both stresses. The rice seedlings treated with 50 mM NaCl, inoculated by Guy11, and pretreated with NaCl for 24 h and then spray inoculated by M. oryzae were pooled for RNA extraction, respectively. Then the transcriptome was analyzed. c Apoplastic polyamine estimation in rice under salt, M. oryzae infection, and both stresses. After 3-d of treatment, polyamine contents (PUT, SPD, and SPM) were measured. Values are means ± SD (n = 3). Significant differences from the control were analyzed by Student’s t-test ** p < 0.01 and * p < 0.05

OsSAMDC overexpression resulted in polyamine accumulation in rice

Sequence analysis revealed that the rice OsSAMDC contains 392 amino acid residues, with a predicted molecular weight of 42.7 KD. By protein BLAST, the SAMDC of Oryza sativa is shown to be 70.30% sequence identical with Triticum aestivum, 68.24% with Saccharum officinarum, 67.09% with Zea mays, and 53.61% with Arabidopsis thaliana. By sequence alignment with other 19 plants, the deduced OsSAMDC protein had two conserved function domains: the proenzyme cleavage site and PEST domain (Additional file 1: Figure S1), which were important in regulating the dynamic balance of various types of polyamines. In order to identify the acting position of the OsSAMDC protein, we fused the OsSAMDC gene onto the N-terminal of GFP and transformed it into Nicotiana benthamiana leaves. The result showed that the OsSAMDC-GFP fusion proteins targeted to the nucleus, cytoplasm, and plasma membrane (Additional file 1: Figure S2).

To explore the potential roles of OsSAMDC in rice immunity, we generated the OsSAMDC-RNAi lines and the OsSAMDC-overexpression lines driven by a constitutive promoter, respectively. We obtained two OsSAMDC knockdown lines (RNAi-2 and RNAi-3) and two OsSAMDC-OX lines (OX-3 and OX-5) by detecting the OsSAMDC gene expression in plants using RT-PCR and RT-qPCR (Additional file 1: Figure S3a–d). Interestingly, the OsSAMDC-RNAi seedlings exhibited reduced root and shoot length than WT; by contrast, the OsSAMDC-OX plants had much longer roots and shoots (Additional file 1: Figure S3e–g), suggesting that OsSAMDC is involved in rice growth and development.

Since OsSAMDC is one of the polyamine biosynthesis enzymes, we therefore tested the total polyamines in OsSAMDC transgenic lines. The levels of all three polyamines (PUT, SPD, and SPM) were significantly increased in the OsSAMDC-OX plants in comparison to the WT (Fig. 3a). The PUT contents increased by 79% and 45%, the SPD contents increased by 78% and 62%, and the SPM contents increased by 71% and 44% in OsSAMDC-OX-3 and -5 plants, respectively (Fig. 3a). In contrast, the three polyamine levels were considerably diminished in OsSAMDC-RNAi lines (Fig. 3b). These results indicate that SAMDC positively regulates the accumulation of PAs in rice leaves.

Fig. 3

OsSAMDC overexpression rice plants accumulated higher contents of PAs. a, b The total polyamine contents in OsSAMDC-OX and OsSAMDC-RNAi plants. Two-week-old transgenic rice seedlings were cultured and total polyamine levels in OsSAMDC-OX and -RNAi plants were measured. FW, fresh weight. Values are means ± SD (n = 3). Different letters indicate the significant differences based on one-way ANOVA with Tukey’s HSD test. c, d The total polyamine contents in OsSAMDC-OX and OsSAMDC-RNAi plants under salt treatment. Rice seedlings were treated with 50 mM NaCl for 5-d. Others aresame as the mentioned above. e, f Growth performances of OsSAMDC-OX and OsSAMDC-RNAi plants under salt-stressed conditions. Seeds were cultured in ¼ MS medium containing 50 mM and 100 mM salt concentration for 7-d. The roots and shoot length (RL and SL) were measured. Values are means ± SD (n = 3). Different letters indicate the significant differences based on one-way ANOVA with Tukey’s HSD test

We also detected the total polyamine levels of these transgenic lines under the salt stress. The OsSAMDC-overexpression rice plants exhibited higher levels of PUT, SPD, and SPM in comparison to WT, whereas the OsSAMDC-RNAi plants displayed decreased polyamine levels (Fig. 3c, d). In addition, under different salt-stressed conditions, the magnitude of enrichment of root length was significantly higher in OsSAMDC overexpression rice seedlings compared with WT, even under high salt (100 mM) conditions (Fig. 3e, f). All these results suggest that SAMDC promoted total polyamine accumulation and plays a key role in rice salt tolerance.

SAMDC-OX transgenic rice plants display enhanced disease resistance while SAMDC-RNAi lines show enhanced disease sensitivity to M. oryzae

To confirm the role of OsSAMDC in blast resistance, we first inoculated the leaves of WT, OsSAMDC-OX, and OsSAMDC-RNAi plants with M. oryzae spores. Both RNAi lines exhibited significantly more disease lesions and fungal biomass than those on the WT plants (Fig. 4a–c), indicating that the RNAi lines are more susceptible to blast fungus. Conversely, the OsSAMDC-OX plant displayed reduced lesions and fungal biomass in comparison to the WT (Fig. 4b, c). In addition, we carried out the punch inoculation assays with the leaves of OsSAMDC transgenic lines. The OsSAMDC-OX lines exhibited considerably smaller lesions in diameter, while the OsSAMDC-RNAi lines displayed larger lesions compared to the wild type (Fig. 4d, e). Collectively, it can be concluded that OsSAMDC exerts a positive regulatory effect on rice resistance to blast fungus.

Fig. 4

OsSAMDC-OX transgenic rice plants displays enhanced disease resistance against M. oryzae. a–c Disease phenotype, lesion number, and relative fungal biomass of OsSAMDC-OX and -RNAi lines upon M. oryzae infection. Two independent lines of OsSAMDC-OX-3 and OX-5, and RNAi-2 and RNAi-3 were used for inoculation assays. Conidial suspensions (1 × 10⁵ conidia/mL in 0.02% Tween-20) were sprayed onto the leaf surface of 2-week-old seedlings for 5-d. Images were taken at 5 dpi. Values are means ± SD (n = 3). Different letters indicate the significant differences based on one-way ANOVA with Tukey’s HSD test. d, e Disease symptoms and lesion length of the OsSAMDC transgenic lines by punch inoculation with M. oryzae conidia. Images were taken at 5 dpi

The apoplastic polyamine levels were elevated in OsSAMDC-OX plants

Because OsSAMDC-OX positively regulates the accumulation of PA in rice leaves (Fig. 3a, c), it is speculated that PAs may contribute to the disease resistance in plants. Thus, we determined the apoplastic PAs in the leaves of OsSAMDC transgenic lines upon M. oryzae infection. During the early time (3 dpi) of M. oryzae infection, all three apoplastic polyamine contents were significantly elevated in OsSAMDC-OX plants compared to WT. The PUT content was increased by 73% and 44% in OX-3 and OX-5 plants, respectively, compared to WT plants. While the SPD levels increased by 50% and 63%, and the SPM contents increased by 64% and 51% (Fig. 5a–c). Similar results were also found at 6 dpi. These results indicated that all three apoplastic polyamine contents were significantly elevated in OsSAMDC-OX plants compared to WT after M. oryzae infection. By contrast, in OsSAMDC-silenced plants, the polyamine contents were observed at significantly low levels when compared to WT at both time points after M. oryzae infection (Fig. 5d–f). These results demonstrated that SAMDC promoted the apoplastic polyamine contents and enhanced disease resistance after M. oryzae infection.

Fig. 5

Apoplastic polyamines increased in OsSAMDC-OX plants. a–c Apoplastic PUT, SPD, and SPM levels in OsSAMDC-OX leaves. Conidial suspensions (1 × 10⁵ conidia/mL in 0.02% Tween-20) were sprayed onto the leaf surface of 2-week-old seedlings, and then apoplastic PA levels were determined at 3 and 6 dpi. Values are means ± SD (n = 3). Different letters indicate the significant differences based on one-way ANOVA with Tukey’s HSD test. d–f Apoplastic PUT, SPD, and SPM levels in OsSAMDC-RNAi plants. Others are same as mentioned above

Salt stressed OsSAMDC-OX plants showed enhanced resistance to M. oryzae

The abovementioned results showed that the OsSAMDC overexpression rice plants accumulated higher levels of polyamines and disease resistance. We further assessed the role of SAMDC in the plant response to salt stress and whether salinity could induce more polyamine biosynthesis and enhanced resistance to rice. Two-week-old WT and transgenic rice seedlings were treated with only Guy11 and another group was pretreated with 50 mM salt, followed by inoculation with M. oryzae conidial suspension. Disease phenotype and PA levels were measured at 5 dpi after fungal treatments. Significantly fewer numbers of lesions (Fig. 6a, b) and fungal biomass (Fig. 6c) were observed in salt treated transgenic rice than Guy11-treated rice. The number of lesions per leaf was significantly reduced by 51% and 43% in overexpression rice under a salt-stressed condition over only Guy11-treated plants (Fig. 6b). In addition, we inoculated M. oryzae conidia in the rice leaf sheath to examine the hyphal growth and colonization efficiency under salt treatment. We found that the fungal hyphae developed faster in infected cells of SAMDC-RNAi lines compared to WT; however, the hyphal growth was restricted in SAMDC-overexpression lines. In consistence with the disease phenotype, salt treatment could inhibit the fungal hyphae growth in transgenic plants. After salt treatment, significantly reduced hyphal growth was observed in SAMDC-overexpression lines (Additional file 1: Figure S4b). Furthermore, the pretreated plants with salt also contain increased amount of all three forms of apoplastic polyamines (Fig. 6d–f). As SAMDC plays a vital role in rice catabolism of polyamine, we observed that PUT, SPD, and SPM contents were higher in OsSAMDC overexpression plants than WT, even though the SPD and SPM content were higher among the three polyamines. Conversely, OsSAMDC-RNAi lines exhibited a decrease in all three polyamine contents when compared to WT (Fig. 6d–f). All these results suggest that salt treatment can enhance the plant immunity by accumulating more polyamines.

Fig. 6

Salt-stressed OsSAMDC-OX plants showed enhanced blast resistance and accumulated more polyamines. a–c Disease phenotype, lesion number, and relative fungal biomass of salt-treated OsSAMDC-OX and -RNAi lines upon M. oryzae infection. Two-week-old rice seedlings were treated with 50 mM NaCl for 24 h. Then the conidial suspension of M. oryzae (1 × 10⁵/mL) was spray-inoculated on the salt-treated rice seedlings. Images were taken at 5 dpi. The fungal biomass was determined by qPCR of M. oryzae Pot2 gene against the rice OsACTIN gene. Different letters indicate the significant differences based on one-way ANOVA with Tukey’s HSD test. Values are means ± SD (n = 3). d–f Apoplastic polyamine contents of OsSAMDC transgenic rice seedlings under salt-stressed condition. Two-week-old rice seedlings were treated with 50 mM NaCl for 24 h. Then the conidial suspension was spray-inoculated on NaCl pretreated rice seedlings and monitored at 3 dpi. FW, fresh weight. Different letters indicate the significant differences based on one-way ANOVA with Tukey’s HSD test. Values are means ± SD (n = 3)

Polyamines inhibit the growth and development of M. oryzae and increase the disease resistance

To confirm the impact of polyamines on the growth and development of M. oryzae, we conducted in vitro tests to assess their performance. Our findings demonstrate that 5 mM SPD and SPM significantly inhibited M. oryzae mycelial growth on complete medium (CM), however, PUT did not affect mycelial growth at both concentrations when compared to control (Fig. 7a, b). Moreover, SPD and SPM significantly suppressed appressorium formation of M. oryzae at concentrations of 1 mM and 5 mM (Fig. 7c). In addition, spore germination, germ tube elongation, and appressorium formation were also inhibited by SPD and SPM at concentrations of 1 mM and 5 mM (Fig. 7d–f).

Fig. 7

Polyamines inhibit the growth and development of M. oryzae and increase disease resistance. a, b Polyamines (SPD and SPM) inhibit the mycelium growth of M. oryzae. 1 mM and 5 mM of PUT, SPD, and SPM were added to the culture medium, respectively. Fresh mycelium was cultured in the medium for 7-d and mycelium growth diameter was measured. Values are mean ± SE. ** indicate the significant difference from control at P < 0.01 by student’s t-test. c Polyamines suppressed the appressorium formation. The conidial (1 × 10⁵/mL) suspension with 0.1, 1, and 5 mM of putrescine, spermidine, and spermine, respectively, were placed on the glass slide. The spores were incubated in a box containing moisture at room temperature, and images were taken at 12 hpi. Scale bars = 5 µm. d–f Estimation of spore development by applying PUT, SPD, and SPM. The conidial suspension supplied with 0.1, 1, and 10 mM of putrescine, spermidine, and spermine was placed on a glass slide separately, and incubated at room temperature for 12 h. Values are mean ± SE. g, h Disease symptoms and relative fungal biomass of the polyamine (SPD and SPM) pretreated WT and OsSAMDC transgenic plants after M. oryzae infection. Two-week-old rice seedlings were sprayed with 1 mM of SPD and SPM for 24 h. Then the conidial suspensions (1 × 10⁵ conidia/mL in 0.02% Tween-20) were sprayed onto the SPD and SPM pretreated rice seedlings. Images were taken at 6 dpi. The fungal biomass was determined by qPCR of M. oryzae Pot2 gene against the rice OsACTIN gene. Values are means ± SD (n = 3). Different letters indicate the significant differences based on 1-way ANOVA with Tukey’s HSD test

Our previous results indicated that rice plants overexpressing OsSAMDC accumulated a higher quantity of endogenous apoplastic PAs, particularly higher levels of SPD and SPM under salt-stressed conditions, which resulted in rice blast resistance. To further explore the involvement of polyamines (SPD and SPM) in rice disease resistance, we first sprayed the SPD (1 mM) and SPM (1 mM) on WT and transgenic plants. We observe that significantly fewer lesions and fungal biomass were observed on polyamine-pretreated SAMDC overexpression plants (Fig. 7g, h). Moreover, after polyamine (SPD and SPM) pretreatment, the silenced transgenic plants showed similar disease phenotype and fungal biomass as WT plants (Fig. 7g, h). This result indicates that exogenously supplied polyamines can markedly subvert the disease susceptibility to M. oryzae in OsSAMDC silenced plants. Collectively, our results suggested that both SPM and SPD could inhibit the growth and development of M. oryzae and increase the disease resistance in rice plants.

Discussion

Plant resistance is significantly improved by the crucial contribution of PAs. Alterations in the inherent levels of polyamines (PAs) or morphological structure of plants have an impact on their ability to withstand abiotic (Tassoni et al. 2008; Alcazar et al. 2010) and biotic stresses (Wimalasekera et al. 2011). Our current results demonstrated that the expression of enzymes responsible for PA biosynthesis was increased following exposure to salt and M. oryzae treatments (Fig. 2b). Salt treatment increased the production of PAs, which may protect the plants from oxidative damage caused by reactive oxygen species (ROS) (Jang et al. 2012). PA accumulation during biotic stress is commonly attributed to the control of PA biosynthesis genes at the transcriptional and translational levels (Walters 2003; Jimenez-Bremont et al. 2014; Tsaniklidis et al. 2020).

In general, when plants suffer from different stresses, the expression of some stress related genes, such as polyamine biosynthesis genes, were induced, which makes the plant more tolerant or resistant. SAMDC is a key enzyme in the regulation of polyamine synthesis and metabolism. Consequently, it has a significant impact on the alteration of endogenous polyamines in plants (Marco et al. 2011). Nevertheless, there are a limited number of reports regarding rice SAMDC, particularly in terms of genetic analysis. We established a research foundation in this study by analyzing and identifying the rice SAMDC gene sequences using bioinformatics, and then investigating the response of the rice SAMDC gene to salt and M. oryzae infection. Several SAMDC homologs have been discovered in some plant species, such as sugarcane (Liu et al. 2010), tall fescue (Wang et al. 2011), and tomato (Liu et al. 2008). Some of these homologs exhibit sequences that closely resemble OsSAMDC. In addition, SAMDC proteins have a characteristic SAMDC protease domain (Additional file 1: Figure S1), which is conserved across several species and likely plays significant roles in plant development and resilience to stress. Liu et al. (2014) demonstrated that the CmSAMDC-GFP fusion protein exhibited localization in the nucleus, cytoplasm, and plasma membrane of melon. Our results proved that the OsSAMDC had a similar localization pattern as the CmSAMDC. As previously mentioned, the SAMDC was implicated in abiotic stress tolerances, including high temperature, salinity, and cold. Therefore, resistant cultivars exhibited higher SAMDC expression than susceptible ones (Groppa and Benavides 2008; Hazarika and Rajam 2011).

We have generated OsSAMDC overexpression and silenced transgenic rice plants and found that SAMDC-OX rice plants accumulated higher levels of polyamines than the SAMDC silenced plants (Fig. 3a, b). Furthermore, the concentration of PUT, SPD, and SPM increased more significantly in overexpression rice plants under salt stress (Fig. 3c, d). Previous study support the observation that the overexpressed BvM14-SAMDC plant accumulated higher amount of SPD and SPM which showed enhanced salt tolerance in sugar beet (Ji et al. 2019). Similarly, down-regulation of SAMDC expression resulted in reduced levels of SPD and SPM and decreased salinity tolerance in transgenic tobacco plants (Moschou et al. 2008). Other research also obtained similar results that a variety of transgenic SAMDC plants exhibit resistance to external stresses across a broad spectrum (Wi et al. 2006).

Although SAMDC-OX plants displayed enhanced rice salt tolerance, we are interested to investigating the responses to pathogen infection. SAMDC-OX transgenic lines showed enhanced disease resistance and accumulated higher PAs after M. oryzae infection (Fig. 6). Our data are consistent with the report that the SAMDC1-overexpressing plants showed an increased tolerance to infection of Pseudomonas syringae and Hyaloperonospora arabidopsidis (Marco et al. 2014). A previous investigation similarly demonstrated that pathogen infection in the inoculated leaves of transgenic lines was significantly diminished when CmSAMDC was over-expressed in Arabidopsis. Furthermore, the increased resistance to powdery mildew seemed to be linked to pathogen-induced cell death (Liu et al. 2014). Previous work reported that exogenous application of polyamine to the medium exhibits antifungal properties, leading to a reduction in the growth of Macrophomina phaseolina, which is responsible for causing disease in soybean seedlings (Santos et al. 2021). Our investigation revealed that two categories of polyamines (SPD and SPM) not only suppressed the growth of mycelium, but also inhibited the germination of spores, elongation of germ tubes, and formation of appressoria in the fungus (Fig. 7d–f), implying that polyamines strongly restrict the growth and development of fungi. We also proved that SAMDC overexpression plants increased the accumulation of SPD and SPM during M. oryzae infection. Our data further revealed that exogenously supplied polyamines could increase the disease resistance (Fig. 7g, h), although SPM had the most significant control effects, followed by SPD over WT. This result suggests that the polyamines, especially SPM and SPD, have antifungal activity against rice blast fungus, which was also confirmed through in vitro testing that 1 mM spermine treatment significantly decreased zoospore production and colony growth of Phytophthora capsica (Koc et al. 2017). Our findings suggest that increased accumulation of polyamines may contribute to the disease resistance in SAMDC-OX plants. Previous studies found that thermospermine, a structural isomer of SPM, triggered plant resistance to P. viridiflava in Arabidopsis through polyamine oxidase (PAO)-mediated thermospermine oxidation (Marina et al. 2013). Other research provided evidence that tobacco plants accumulated SPM in response to infection by the (hemi)biotrophic bacterial pathogen P. syringae pv. tabaci that leads to the activation of defense-related genes and defense responses, ultimately resulting in increased tolerance to the disease (Moschou et al. 2009). Even, exogenous SPM increases the disease resistance of Arabidopsis against P. viridiflava, which is compromised by the PAO inhibitor (Gonzalez et al. 2011). According to our findings, genes involved in polyamine biosynthesis are activated in response to external stresses and serve as a defensive function.

Conclusions

Salt stress has complex effects on plants, and its impact on plant disease resistance is influenced by various factors. Generally, salt stress is known to adversely affect plant growth and development. However, studies suggest that moderate levels of salt stress can induce certain defense in plants, which potentially leads to enhanced disease resistance. Based on our results, we hypothesized that after the salt (NaCl) pretreatment, the microenvironment of the plant’s intercellular space was unfavorable for colonization of the pathogenic microorganisms. This may be due to the changes in the physiology of the plant to adapt to the saline environment and the PTI response as well. Therefore, this study provides additional evidence for the role of polyamines in plant response to biotic stress and supports the hypothesis that SAMDC-mediated polyamines may exert their protective action through transcriptional changes in defense genes, particularly in response to apoplastically-localized plant pathogens (Fig. 8). Further research is needed to understand the underlying mechanisms and to develop strategies for managing salt stress in agriculture while maintaining plant disease resistance.

Fig. 8

Overview of responses induced by salt stress and pathogen infection in rice plants. OsSAMDC, an important polyamine biosynthesis gene, was induced during salt stress and M. oryzae infection. In the salt-stressed plant, more polyamines are accumulated in the apoplast. Polyamine (PUT, SPD, and SPM) biosynthesis responds to apoplast-localized pathogens, ultimately leading to antimicrobial defense responses

Methods

Plant and fungal strain growth conditions

Rice (Oryza sativa subsp. Japonica cv Nipponbare) was used as the wild type (WT) in this study. The OsSAMDC (Os09g25625) coding sequence was PCR amplified and cloned into the pCAMBIA1390 binary vector with an Ubi promoter to generate an overexpression vector. To generate OsSAMDC-RNAi constructs for gene suppression, 300–500 bp fragments of gene sequences are generated by PCR from the Os09g25625 gene and the resulting PCR fragments were cloned into the Gateway pENTR/D-TOPO cloning vector as described before (Miki and Shimamoto 2004). All the vectors were introduced into Agrobacterium tumefaciens strain EHA105α through electroporation, and the resulting strains were used for transformation as described previously (Xiong et al. 2017). All primers used in this experiment are shown in Additional file 2: Table S1. Seeds were surface sterilized with 70% ethanol for 5 min, 30% sodium hypochloride for 20 min. The plants were cultivated under controlled conditions at a temperature of 28 °C, with a photoperiod of 16 h of light and 8 h of darkness and a relative humidity of 75%. Magnaporthe oryzae strain Guy11 was grown on oatmeal agar medium (30 g/L oat, 0.6 g/L calcium carbonate, and 16 g/L agar) for approximately 10-d. After scraping the aerial mycelium from the plate, the conidia were induced under white light for a duration of 2-d. The spores were subsequently collected in sterile water.

RNA extraction, RT-PCR, and RT-qPCR assays

The rice leaves were harvested at indicated times. TRIZOL (Invitrogen, USA) reagent was utilized to extract total RNA. The cDNA synthesis was performed using HiScript QRT Super Mix according to the manufacturer’s instructions (Vazyme, China). Reverse transcription (RT)-PCR was performed according to the manufacturer’s instructions using Primescript reagent kit with the gDNA Eraser (TaKaRa, Kusatsu, Japan). ChamQ SYBR qPCR Master Mix (Vazyme, China) was used for RT-qPCR analysis with CFX96TM Real-time System (Bio-RAD, USA). OsACTIN was employed as a reference gene to standardize all qRT-PCR data. The relative expression was calculated using the 2^−ΔΔCt method. The primers used are listed in Additional file 2: Table S1.

RNA-seq analysis

In order to conduct RNA-seq analysis, total RNA was extracted using TRIZOL reagent from the rice seedlings that treated with 50 mM NaCl, and spray inoculated by M. oryzae for 24 hpi. Library preparation and sequencing processes were described previously (Yang et al. 2017; Meng et al. 2020). Briefly, RNA integrity was evaluated using a Bioanalyzer 2100 (Agilent Technologies, USA). The libraries were constructed and sequenced on an Illumina NovaSeq 6000 at Novegene (Beijing) and 150 bp paired-end reads were generated. Raw reads of fastq format were firstly processed by fastp to generate clean reads (Chen et al. 2018). The trimmed reads were aligned against the O. sativa genome (release 7 of the MSU) using Hisat2 (Kim et al. 2019), followed by featureCounts (Liao et al. 2014) to generate a count matrix. Differentially expressed genes were detected by DESeq2 (Love et al. 2014). DEGs were defined by a cut-off of |logFC|> 1, p-adjust < 0.05 as criteria.

Sequence analysis of rice SAMDC

To study the evolutionary relationship of SAMDC between rice and other plant species, the OsSAMDC gene sequence and SAMDC protein sequence of 20 plants including Arabidopsis, wheat, tomato, and carrot were obtained by searching the National Center for Biotechnology Information (NCBI) Genome Database. The conserved motifs of different species and the conserved domains of SAMDC protein sequences were searched by the online MEME tool (https://meme-suite.org/meme/) and NCBI CDD (https://www.ncbi.nlm.nih.gov/cdd/), and visualized by TB Tools (Chen et al. 2020). In addition, the multiple sequences of twenty plants were aligned with Clustal X and Genedoc, and the phylogenetic tree was constructed with MEGA 5.1 by the Neighbor-Joining method.

Subcellular localization of OsSAMDC

The OsSAMDC was combined with the N-terminal of green fluorescent protein (GFP) to yield a fusion protein. The entire coding region of the OsSAMDC gene amplified by PCR was inserted into the Xho I and Kpn I sites of vector pCAMBIA1300-GFP. The fusion construct was transformed into A. tumefaciens strain EH105α and the bacterial suspension (OD₆₀₀ = 0.5) was infiltrated into N. benthamiana leaves by the injection infection method (Zhou et al. 2018). N. benthamiana leaves trans`formed with p35S:GFP were used as controls. Fluorescence was examined under a two-photon laser confocal microscope (Zeiss LSM880) 2-d after transformation.

Plant infection assays

The plant infection assay was conducted using the methodology described by Yang et al. (2017). For spray inoculation, conidial suspensions were adjusted to 1 × 10⁵ conidia/mL in 0.02% (v/v) Tween-20. Then, the suspension was sprayed uniformly onto the rice leaf surface of 2-week-old rice seedlings from all directions. Subsequently, the plants were placed in a growth chamber at 28°C without light for 20 h, then switched back to normal growth conditions. For punch inoculation, a 10 μL volume of spore suspension was applied to the punched leaves. Photographs were taken at 5-d after inoculation. The fungal biomass was determined by qPCR using specific primers for the Pot2 gene of M. oryzae and normalized to the reference rice gene, OsACTIN (Qi and Yang 2002). The primers used are listed in Additional file 2: Table S1.

Apoplast and total PAs quantification

The polyamines from the rice apoplast, was performed by modification of the method described by Yoda et al. (2003). In brief, 2 ~ 3 g of leaves was cut (4–5 cm), weighed and surface washed with ddH₂O. Then, leaves were kept in PBST buffer solution (pH 7.2, NaCl 8 g/L, KCl 0.2g/L, Na₂HPO₄.12H₂O 1.44 g/L, KH₂PO₄ 0.24 g/L, and Tween-20 0.01%) for washing several times and the air was eliminated from the leaves by vacuum pump for 10 to 12 min. Subsequently, they were subjected to centrifugation (1000 g for 7 min at 4°C) to recover apoplastic fluid by placing them in a 10-mL syringe, which was set in a 50-mL Falcon tube. Extracted fluid-containing polyamines were derivatized with benzoyl chloride as described (Flores and Galston 1982). In brief, 100 μL of extracted apoplastic fluid was taken into 1.5 mL eppendorf tube, then 1 mL of 5% TCA (trichloroacetic acid) was added, and the tube was kept in the dark for 1 h at 4°C. After centrifugation at 12,000 g at 4°C for 30 min, 500 μL of supernatant was collected in a new 15 mL falcon tube. The supernatant was added to 7 μL of benzoyl chloride and 1 mL of 2 M NaOH and vortexed for 30 s. After incubation at 37°C for 30 min, 2 mL of saturated NaCl and 2 mL of ethyl ether were added to it and centrifuged at 1500 g for 15 min at 4°C. 1 mL of the ether phase was collected in a new 1.5 mL eppendorf tube and dried under nitrogen gas. The residue was dissolved in 150 μL of HPLC-grade methanol. Similarly, the reaction mixture was prepared as the standard samples, containing 0.2, 0.4, 0.6, 0.8, and 1.0 mmol/L of individual PAs.

For extraction of total polyamines (PUT, SPD, and SPM) from rice leaves were conducted using a modified method described by Zapata et al. (2003). The leaf samples (100–200 mg) were homogenized with liquid nitrogen using 2 iron balls and extracted in 1 mL of 5% TCA (trichloroacetic acid) for 1 h at 4°C. The supernatant (500 μL) was transferred to a fresh 15 mL tube after 30 min of centrifugation at 12,000 g and 4°C. Benzoyl chloride (7 μL) and 2 M NaOH (1 mL) were added to the supernatant, and the mixture was vortexed for 20 s. Following 20 min of incubation at 37 °C, 2 mL of saturated NaCl and 2 mL of ethyl ether were added to it. The mixture was then centrifuged at 1500 g for 10 min at 4°C. 1 mL of the ether phase was then transferred to a new tube and dried using a vacuum. The residue was dissolved in 150 μL of HPLC grade methanol. HPLC analysis was performed with an ODS-BP column (5 μm, 4.6 mm × 200 mm, Elite, China) on a system consisting of a ddH₂O pump-A and a gradient (acetonitrile) pump-B, and a UV detector. 20 μL samples were injected and polyamines were eluted with a gradient (acetonitrile) 50% and ddH₂O 50% at a flow rate of 1 mL/min. The benzoyl-polyamines were detected by absorbance at 254 nm. The results are expressed as nmols per gram of fresh weight (nmol/g FW) and are the mean ± SE of extractions from three different samples per treatment.

Fungal cell development and polyamine treatments in in vitro

Spore germination, germ tubes elongation, and appressorium formation were examined according to the method described by Ahn et al. (2003) and Lee and Dean (1993). Briefly, a 50 μL drop of 1 × 10⁵/mL concentration of conidial suspension with 0.1, 1, and 5 mM of putrescine (PUT), spermidine (SPD), and spermine (SPM) was placed on a glass slide (CITOTEST-Adhesion microscope slides; 25 × 75 mm, 1 mm–1.2 mm), and sealed in a box containing moisture. The spores were incubated at room temperature for 12 h. The percentage of spore germination, germ tube elongation, and appressorium formation were examined on a plastic coverslip (avantor VWR; 22 × 22 mm) at 12 h by a microscope at least 100 conidia per replicate. Five mycelial disks were transferred from actively growing colonies of M. oryzae (Guy11) on complete medium to freshly prepared solid complete medium (Casein acid hydrolysate 3g/L, Casein enzymatic hydrolysate 3g/L, Yeast extract 6g/L, Sucrose 10g/L, and agar 15g/L) containing 1mM and 5 mM of putrescine, spermidine, and spermine and finally incubated at 28°C for 7 days, and the diameter of mycelia was measured.

Polyamine pretreatments and salt treatments

M. oryzae conidial suspension (1 × 10⁵ spores/mL concentration with 0.02% Tween-20) was sprayed 24 h later after the pretreatments with 1 mM SPD and SPM onto 2-week-old rice seedlings. The disease phenotype was determined at 6 dpi. Inoculated plants were grown in the growth chamber under the same conditions as described before. For salt treatment, the same concentration of spores was sprayed 1 day before salt treatment. 50 mM salts were used to treat the 2-week-old rice plants. The store conditions, polyamine extraction and measurement were described in the earlier section.

Statistical analysis

All the data were analyzed by single factor analysis of variance (ANOVA) and Student’s t-tests using IBM SPSS 26.0 software (SPSS Inc., Chicago, IL, USA). Duncan’s multiple comparison method was used to analyze the difference between different treatments at a P < 0.01–0.05 level of significance.