Osa-miR11117 Targets OsPAO4 to Regulate Rice Immunity against the Blast Fungus Magnaporthe oryzae

Abstract

The intricate regulatory process governing rice immunity against the blast fungus Magnaporthe oryzae remains a central focus in plant–pathogen interactions. In this study, we investigated the important role of Osa-miR11117, an intergenic microRNA, in regulating rice defense mechanisms. Stem-loop qRT-PCR analysis showed that Osa-miR11117 is responsive to M. oryzae infection, and overexpression of Osa-miR11117 compromises blast resistance. Green fluorescent protein (GFP)-based reporter assay indicated OsPAO4 is one direct target of Osa-miR11117. Furthermore, qRT-PCR analysis showed that OsPAO4 reacts to M. oryzae infection and polyamine (PA) treatment. In addition, OsPAO4 regulates rice resistance to M. oryzae through the regulation of PA accumulation and the expression of the ethylene (ETH) signaling genes. Taken together, these results suggest that Osa-miR11117 is targeting OsPAO4 to regulate blast resistance by adjusting PA metabolism and ETH signaling pathways.

1. Introduction

Pathogens such as bacteria, fungi, and oomycetes frequently attack plants, prompting the plants to evolve a sophisticated defense system comprising pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity (ETI) to fight against the invaders [1]. Accumulating evidence has revealed that plant microRNAs (miRNAs) are critical regulators in both PTI and ETI responses against pathogens [2,3]. miRNAs are a type of endogenous RNA (21~24 nucleotides) that consist of a single strand and play a crucial role in regulating gene expression at the post-transcriptional level or the translation level [4]. Importantly, miRNAs play a pivotal role as significant regulators of diverse cellular and biological processes, such as plant growth, development, reproduction, and responses to both biotic and abiotic stresses [5,6]. Under the invasion of pathogens, miRNA expression in some plants changed significantly. Differentially expressed miRNAs may play an important role in plant disease resistance by inhibiting or activating the expression of some target genes, regulating plant signal transduction pathways, and activating key factors of the disease resistance pathway [7,8]. The precise function of a given miRNA depends on the specific target genes and their involvement in various biological processes. For example, miR393, the first discovered miRNA in Arabidopsis, regulates the host immunity against Pseudomonas syringae DC3000 through inhibiting auxin signaling by targeting the auxin receptors, including TIR1, AFB2, and AFB3 [9]. Furthermore, it has been identified that miR160a, miR398b, and miR773 play a crucial role in the modulation of callose deposition in PTI signaling [10]. In addition, the miR482/miR2118/miR472 superfamily possesses the ability to regulate the expression of NBS-LRR genes to prevent the excessive expression of these genes when there is no pathogen present, indicating the critical roles of miRNA in ETI signaling [11,12].

Polyamines (PAs) are a group of amine molecules, mainly including putrescine (Put), spermidine (Spd), spermine (Spm), and thermospermine (T-Spm), that exist widely across various living organisms. There is evidence pointing to the significant role of PAs in the interactions between plants and pathogens [13]. The levels of PAs and the activity of enzymes involved in PA metabolism were observed to increase upon infection of plant tissues by both biotrophic and necrotrophic pathogens [14,15]. Reduced Put levels in adc loss-of-function mutants and adc-silenced lines lead to increased susceptibility to pathogens [16,17]. Conversely, the overexpression of the AtADC2 gene or supplementation of Put in Arabidopsis induces the expression of plant defense genes and promotes local salicylic acid biosynthesis [16]. In addition to Put, Spm is also regarded as a significant signaling molecule that regulates resistance to bacterial pathogens and viruses [11,18]. Similarly, reduced T-Spm (a structural isomer of Spm) levels in cotton acl5-silenced plants lead to susceptibility to Verticillium dahliae infection [19]. Moreover, the pathogenic bacterium Ralstonia solanacearum produces Brg11 effector protein to target the tomato ADC1 and ADC2 genes and promote Put accumulation [20]. In response to plant pathogens such as P. syringae, there is an increase in polyamine oxidase (PAO) activity, leading to Spm secretion and H₂O₂ accumulation in the apoplast, which contributes to the hypersensitive response (HR) [21]. In tobacco, inhibiting the expression of the gene coding for apoplastic PAO prevented PA degradation, cryptogein-induced apoplastic H₂O₂ generation, and cell death [22]. In addition to its cytotoxic effect, PAO-generated H₂O₂ also has a signaling role in the HR [22]. Although the enzyme characteristics of all Arabidopsis PAOs and most rice PAOs have been determined, their biological functions in plant defense mechanisms against pathogens still remain ambiguous.

Rice is a crucial food source for more than half of the global population, but its production is often threatened by a variety of diseases, including rice blast caused by M. oryzae. In recent years, miRNAs have been identified as key regulators during rice responsive to M. oryzae infection. For instance, miR398b was found to enhance rice immunity against M. oryzae by targeting multiple SOD genes to boost H₂O₂ accumulation [23]. In contrast, miR164a [24], Osa-miR167d [25], miR168 [26], miR169 [27], miR319b [28], miR396 [29], miR444b.2 [30], miR530 [31], Osa-miR535 [32], miR1432 [33], Osa-miR1871 [34], and Osa-miR1873 [35] exert a negative regulatory influence on rice’s immune response to M. oryzae. Conversely, Osa-miR159a [36], miR160a [37], Osa-miR162a [38], miR166k-miR166h [39], and miR7695 [40] have been identified as contributors to enhancing rice immunity against M. oryzae.

In our previous study, we identified a number of miRNAs that were differentially responsive to M. oryzae [41]. Among them, Osa-miR11117 (previously named miR-T21) was differentially accumulated in the susceptible and resistant rice accessions upon M. oryzae infection, indicating Osa-miR11117 might be involved in the regulation of rice immunity against M. oryzae. In this study, we explored Osa-miR11117 and its target OsPAO4 in rice defense against M. oryzae. First, Osa-miR11117 responded to M. oryzae and displayed varying patterns in susceptible and resistant rice lines. Overexpression of Osa-miR11117 increased susceptibility, while reduced expression led to some resistance. Second, OsPAO4, identified as a target gene of Osa-miR11117, played a crucial role in rice’s defense. Third, OsPAO4 responded to Spm and Spd, hinting at its involvement in PA catabolism. Last, knocking out OsPAO4 made rice more susceptible, while overexpression of OsPAO4 enhanced resistance and impacted polyamine levels and the ethylene signaling pathway. This study highlights that Osa-miR11117 targets OsPAO4s to modulate rice blast resistance via polyamine metabolism and ethylene pathways.

2. Results

2.1. Osa-miR11117 Is Responsive to Blast Fungus

In earlier investigations, we identified several novel miRNA candidates that were responsive to M. oryzae in rice [41]. Herein, we focused on one such miRNA candidate, miR-T21, in Dong et al. [41] and conducted further investigations. Upon searching in miRBase, it was observed that the mature sequence of miR-T21 is relatively conserved, showing significant homology with miRNAs from various species, including lja-miR11117a-3p/lja-miR11117b-3p from lotus, hsv2-miR-H6-3p from herpesvirus, gra-miR7494b from canary, and hsa-miR-5196-3p from humans (Figure S1). Due to the high similarity of miR-T21 with lja-miR11117a-3p/lja-miR11117b-3p, herein, we rename ‘miR-T21’ as ‘Osa-miR11117’. Osa-miR11117 is a 23-base intergenic miRNA located on the positive strand at the end of the long arm of rice chromosome 2, between the genes LOC_Os02g35840 and LOC_Os02g35860 (Figure 1A). UNAFold analysis [42] predicted that it could be folded into a typical miRNA precursor stem-loop structure (Figure 1B). The expression of Osa-miR11117 was confirmed in resistant accession (Pik H4 NIL) by Northern blot (Figure 1C). Analysis of the Osa-miR11117 GUS promoter activity indicates that it is mainly expressed in the coleoptile and leaves, with low expression in the young roots, but not in the stems and internodes (Figure 1D), indicating the expression of Osa-miR11117 is tissue-specific. Furthermore, using stem-loop qRT-PCR analysis, it was found that Osa-miR11117 displays a different expression pattern in the susceptible (LTH) and resistant accessions in response to blast fungus infection (Figure 1E). Upon M. oryzae invasion, the amounts of Osa-miR11117 were significantly increased at 6 h and 72 h post-inoculation (hpi) in LTH but kept unchanged at 12–48 hpi (Figure 1E). In contrast, the accumulation of Osa-miR11117 was increased dramatically at 6 and 24 hpi in Pik H4 NIL but decreased at 12, 48, and 72 hpi (Figure 1E). These results indicated that Osa-miR11117 is responsive to M. oryzae infection and functions in rice immunity to blast fungus.

2.2. Overexpression of Osa-miR11117 Enhances Susceptibility to Infection by M. oryzae

To investigate the impact of Osa-miR11117 on rice blast resistance, we generated transgenic lines with increased expression of the miRNA (OX11117) and the target mimic of the miRNA (MIM11117), and CRISPR/Cas9-mediated mutagenesis of Osa-miR11117 (KO11117) under the Pik-H4 NIL background, respectively. Three lines of each transgenic line (MIM11117 and KO11117) that showed decreased expression of Osa-miR11117 and OX11117 that showed increased expression of Osa-miR11117 were selected for subsequent analysis (Figure 2A). Next, we examined whether the overexpression or knockout of Osa-miR11117 has a consequence in blast disease resistance. Two-week-old leaves of OX11117, MIM11117, KO11117, and wild-type plants were punch-inoculated with M. oryzae GDYJ7 spores, and disease symptoms were followed over time. Compared with wild-type plants, OX11117 plants consistently exhibited higher susceptibility to infection by M. oryzae, as evidenced by disease lesions and relative fungal biomass compared to the control, while MIM11117 and KO11117 lines showed a little resistance to M. oryzae (Figure 2B–D). These findings suggest that overexpression of Osa-miR11117 can compromise blast resistance.

2.3. OsPAO4 Is One of the Target Genes of Osa-miR11117

To identify the target genes of Osa-miR11117, we used the online tool psRNATarget website (https://bio.tools/psrnatarget accessed on 12 March 2019) and predicted the eight most potential targets with strict parameters (Table S1). To further examine which predicted genes are the target genes of Osa-miR11117, a green fluorescent protein (GFP)-based reporter assay in Nicotiana benthamiana plants was performed. Briefly, two constructs, including 35S: target gene-GFP and 35S: Osa-miR11117, were coinjected into N. benthamiana leaves for further observation. After examining these candidate genes with the GFP-based reporter assay, the protein expression of two candidate genes (LOC_Os04g57550 and LOC_Os05g51380) was inhibited by Osa-miR11117 (Figure S2). LOC_Os04g57550, encoding the polyamine oxidase 4 (OsPAO4) in rice, was selected for further study. To further verify that Osa-miR11117 indeed downregulates the expression of OsPAO4, we conducted the GFP-based reporter assay with two constructs, including 35S:OsPAO4ts-GFP and 35S:OsPAO4mts-GFP. The former two contained the putative target site of OsPAO4 and a mutated target site, respectively (Figure 3A,B). Coexpression of Osa-miR11117 and 35S:OsPAO4ts-GFP resulted in a noticeable reduction in GFP intensity and protein concentration in N. benthamiana leaves compared to expression of the construct alone (Figure 3C,D). In contrast, coexpression of 35S:OsPAO4ts-GFP alone or 35S:OsPAO4mts-GFP with Osa-miR11117 did not lead to any changes in GFP expression or protein concentration (Figure 3C,D). The expression level of OsPAO4 in the transgenic lines (OX11117, MIM11117, and KO11117) was further validated. Compared to the wild type, OsPAO4 is significantly enhanced in both KO11117 and MIM11117 transgenic plants (Figure 3E). Interestingly, in OX11117 transgenic plants, the expression of OsPAO4 is also highly increased (Figure 3E), strongly suggesting that OsPAO4 is very likely a target gene of Osa-miR11117, and Osa-miR11117 might inhibit the expression of OsPAO4 at translation level.

2.4. OsPAO4 Is One M. oryzae and PA Responsive PAO Gene

To further explore the involvement of OsPAO4 in rice blast resistance, we examined the amounts of OsPAO4 in the susceptible accession (LTH) and the resistant accession (Pik-H4 NIL). Pik-H4 NIL carries a resistance (R) gene Pik-H4 and exhibits resistance to M. oryzae isolates (GDYJ7) carrying Avr-PikE. After inoculating GDYJ7 on LTH and Pik-H4 NIL leaves, the abundance of OsPAO4 decreased first (6 and 12 hpi) and increased after 24 hpi in LTH (Figure 4A). Pik-H4 NIL also decreased at 6 and 12 hpi but increased more at 48 and 72 hpi compared to LTH, which is a little contrary to the expression level of Osa-miR11117 in response to M. oryzae infection (Figure 1E), suggesting the involvement of OsPAO4, a target of Osa-miR11117, in the rice blast resistance process.

There are seven PAO genes in rice [43]. Among them, OsPAO4 is closed to OsPAO5, and they are both closed to AtPAO4 (Figure S3A). Converse to OsPAO2, OsPAO6, and OsPAO7, the other four PAO genes (OsPAO1, OsPAO3, OsPAO4, and OsPAO5) expressed in a higher level in rice leaves (Figure S3B). Interestingly, only OsPAO4 is responsive to M. oryzae (Figure 4A and Figure S3C). Further, semi-quantitative RT-PCR and qRT-PCR assay indicated that OsPAO4 expresses a high level in callus and root, indicating the important roles of OsPAO4 in rice growth and development (Figure S4A,B).

It has shown that the products of OsPAO3, OsPAO4, and OsPAO5 localize to peroxisomes, and they catalyze the polyamine back-conversion reaction [44]. The role of OsPAO4 expression in response to PAs was further examined. As shown in Figure 4B, PAs treatment did not significantly affect the expression of OsPAO4 in the susceptible accession. Interestingly, after PAs treatment, both Spm and Spd significantly increased the expression of OsPAO4, while Put did not, in resistant accession (Figure 4C). These results indicated that OsPAO4 may be involved in rice blast resistance through the Spm and/or Spd catabolism pathway in blast-resistant accession.

2.5. Knockout of OsPAO4 Enhances the Susceptibility to Infection by M. oryzae

To investigate the involvement of OsPAO4 in rice blast resistance, ko-ospao4 transgenic lines were generated under Pik-H4 NIL background using CRISPR/Cas9 gene editing technology. Three lines with different mutation sites were selected for further study. The ko-ospao4 lines enhanced the susceptibility to infection by M. oryzae with larger lesions (Figure 5A,B) and more fungal biomass than the WT control by punch methods (Figure 5C). Further, qRT-PCR assay showed that the expression of PR1a, PR1b, and PR10a was significantly reduced in the ko-ospao4 transgenic lines (Figure 5D), which is consistent with their decreased disease resistance. We then measured H₂O₂ contents and the activities of the H₂O₂-scavenging enzymes, such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in WT and ko-ospao4 lines. The ko-ospao4 lines had significantly lower H₂O₂ contents than the wild type (Figure 5E), indicating that less H₂O₂ accumulated in ko-ospao4 lines. In addition, SOD, POD, and CAT activities were significantly higher in the ko-ospao4 lines than in the wild type (Figure 5F–H), indicating that the active oxygen scavenging ability was enhanced in ko-ospao4 lines. These results indicate that less ROS accumulated in ko-ospao4 leaves than in the wild type. H₂O₂ contents of ko-ospao4 and WT leaves were further measured after inoculation by M. oryzae. Compared to the wild type, H₂O₂ contents were significantly reduced in ko-ospao4 leaves (Figure 5I). These above findings suggest that the knockout of OsPAO4 enhances the enzyme activity in general and weakens the disease resistance of rice to blast fungus.

2.6. Overexpression of OsPAO4 Enhances the Resistance against M. oryzae

To further investigate whether OsPAO4 positively contributes to rice blast resistance, OsPAO4 over-expressing transgenic plants were generated under Pik-H4 NIL background, detached leaves from OsPAO4 over-expressing plants were challenged by punching inoculation with Guy11 (a compatible strain to Pik-H4 NIL line) spores. The leaves of OsPAO4 over-expressing plants developed mild disease symptoms (Figure 6A), manifested by significantly smaller lesion sizes (Figure 6B) and less fungal biomass on transgenic leaves compared to that from WT plants (Figure 6C), which is inconsistent with further inoculation assay using spray methods (Figure 6D). As opposite to ko-ospao4 transgenic lines (Figure 5D), the expression of PR1a, PR1b, and PR10a was significantly increased in OsPAO4 over-expressing plants (Figure 6E), which is consistent with the enhanced disease resistance (Figure 6A–C). As expected, the OE-OsPAO4 lines exhibited notably elevated H₂O₂ content in comparison to the wild type (Figure 6F). Furthermore, the activities of SOD, POD, and CAT were substantially diminished in the OE-OsPAO4 lines as opposed to the wild type (Figure 6G–I). Hence, these collective observations strongly imply that overexpression of OsPAO4 can improve rice blast resistance by regulating the level of H₂O₂.

2.7. OsPAO4 Regulates Rice Blast Resistance by Controlling Both the Accumulation of PA and the Ethylene Signaling Pathway

Due to its potential role in regulating the levels of H₂O₂ and its contribution to rice blast disease resistance, the function of OsPAO4 in PA accumulation was investigated in blast-resistant accession (Pik-H4 NIL). As shown in Figure 7A–C, knockout of OsPAO4 or overexpression of OsPAO4 can both lead to accumulation of Spm and Spd, but not the Put. Furthermore, knockout of OsPAO4 resulted in a significantly higher accumulation of Spd and Spm compared to the wild type, suggesting that changes in OsPAO4 expression levels affected polyamine accumulation, closely tied to its terminal metabolic function. Additionally, post-inoculation with the rice blast pathogen, Spd in the knockout mutant rapidly degraded, maintaining relative stability at 12 h and 24 h, while Spm consistently decreased (Figure 7A,B). OsPAO4 knockout mutants became more susceptible (Figure 5A–C), indicating Spm’s close association with maintaining rice blast disease resistance. Overexpression of OsPAO4 led to a trend of initially decreasing followed by increasing levels of Spm and Spd after pathogen inoculation, suggesting their important roles in the immune response. These results indicate that both knockout and overexpression of OsPAO4 can lead to the accumulation of Spd and Spm, which are responsive to blast fungus invasion.

Since PAs share a common precursor, S-adenosyl-L-methionine (SAM), with ethylene (ETH), alterations in polyamine levels could impact ethylene synthesis and metabolism, thereby regulating the immune response. Consequently, we conducted fluorescence quantitative analysis of genes related to the ethylene synthesis and metabolism pathways. The results indicate that the knockout of OsPAO4 resulted in an increase in the expression level of the transcriptional repressor OsERF3, a decrease in ethylene production, and increased susceptibility (Figure 5A and Figure 7D). This corresponds to the downregulation of OsACO2, OsACO3, and OsACS1 gene expression (Figure 7E). Notably, the expression of OsACO1 was accumulated significantly in OsPAO4 knockout lines, and the expression of OsACO3 was inhibited in both OsPAO4 knockout and over-expressing lines. These results indicate that the balance of OsPAO4 expression is very important in rice resistance to M. oryzae through the regulation of PA accumulation and the ETH pathway.

3. Discussion

miRNAs, serving as either positive or negative regulators, play crucial roles in the interactions between plants and pathogens [2]. In rice, numerous miRNAs have been discovered to respond to M. oryzae, such as miR160a [37], miR164a [24], miR166k-miR166h [39], Osa-miR167d [25], miR169 [27], miR319b [28], miR396 [29], miR398b [23], Osa-miR439 [45], miR444b.2 [30], Osa-miR535 [32], and miR7695 [40], which have been identified as positive or negative regulators. In our previous study, we obtained 52 novel miRNAs related to rice blast fungus response from the constructed small RNA sequencing library, and degradome sequencing was used for further target gene determination for these novel miRNAs [41]. Among these novel miRNAs, Osa-miR11117 (previously named miR-T21) was selected for further characterization of its role in the regulation of rice blast resistance.

3.1. Impact of Osa-miR11117 on Rice Blast Resistance

Osa-miR11117 is a type of miRNA consisting of 23 bases, and its expression is found in high levels in coleoptile and leaf tissues but is comparatively lower in the stem and internode tissues compared to the roots (Figure 1A–D). This indicates that Osa-miR11117 exhibits tissue-specific expression. The responsiveness of Osa-miR11117 to M. oryzae invasion is different between susceptible and resistant accessions. This observation is similar to previous research findings such as Osa-miR439 [45] and Osa-miR167d [25], indicating that Osa-miR11117 is differentially responsive to M. oryzae. Through multiple evidence by using overexpression, target mimicry, and gene knockout of Osa-miR11117 transgenic lines, we confirmed that Osa-miR11117 negatively regulates rice blast resistance (Figure 2B–D). These findings suggest that Osa-miR11117 is highly responsive to M. oryzae infection and plays a significant role in the immunity of rice against the blast fungus. This responsiveness, combined with its tissue-specific expression, makes Osa-miR11117 a compelling candidate in the intricate network of rice defense mechanisms.

3.2. OsPAO4 Is a Direct Target of Osa-miR11117

Through bioinformatics prediction and the examination of the expression level of the candidate target genes in N. benthamiana leaves (Table S1), LOC_Os04g57550.1 (OsPAO4) and LOC_Os05g51380.2 emerged as potential target genes. Indeed, the findings presented in Figure S2 suggest that LOC_Os05g51380.2, encoding a member of the cysteine protease family proteins, could potentially be another target gene of Osa-miR11117. It is well-established that miRNAs often operate by regulating the expression of multiple target genes to fine-tune different pathways [46]. Therefore, the role of LOC_Os05g51380.2 needs further investigation and examination. Herein, by utilizing the GFP-based reporter assay in N. benthamiana plants, we found the expression of OsPAO4 (OsPAO4mts-GFP) with mutated target sites cannot be inhibited, whereas the expression of OsPAO4 (OsPAO4ts-GFP) without mutations can be suppressed (Figure 4B,C), which were further confirmed with the western blot assay (Figure 4D), strongly indicating that Osa-miR11117 is a regulator of OsPAO4’s expression. Interestingly, in both the susceptible and resistant accessions, Osa-miR11117 and OsPAO4 exhibited contrasting expression patterns in response to M. oryzae infection (Figure 1E and Figure 4A). Similar regulatory mechanisms have been elucidated in other miRNAs; for example, miR396 negatively regulates rice blast disease resistance via suppressing multiple OsGRFs, which in turn differentially control growth and yield [29]. In addition, Osa-miR1432 confers rice bacterial blight resistance by suppressing OsCaML2 [47]. The ghr-miR5272a-mediated regulation of GhMKK6 expression contributes to the immune response in cotton [48]. These reports and our results illustrate that miRNAs are widely present in the regulation of plant disease resistance and participate in plant disease resistance pathways by controlling the expression levels of target genes.

3.3. OsPAO4 Positively Regulates Rice Blast Resistance

Among the seven PAO genes in rice, OsPAO4 is closed to OsPAO5, and they are both closed to AtPAO4 (Figure S3A). OsPAO5 has been previously shown to have a detrimental effect on the lengthening of the mesocotyl by releasing less H₂O₂ and synthesizing more ethylene, which allows for the development of directly planted rice varieties that exhibit enhanced crop yield [43]. The four PAO genes (OsPAO1, OsPAO3, OsPAO4, and OsPAO5) expressed a higher level in rice leaves, and qRT-PCR analysis showed only OsPAO4 responded to M. oryzae infection in rice leaves (Figure 4A and Figure S3C), indicating the important roles of OsPAO4 in blast resistance regulation. Further examination of OsPAO4 expression in response to PA treatment showed, except Put, both Spm and Spd led to an increase in the expression of OsPAO4 in the blast-resistant accession (Figure 4B,C), which is in accordance with the role of OsPAO4 in Spm to Spd back-conversion processes and in PA catabolism.

Knocking out OsPAO4 increased susceptibility to M. oryzae compared to the wild-type control (Figure 5A–C) and upregulated PR genes (PR1a, PR1b, and PR10a) (Figure 5D), suggesting a potential link between R gene Pik-H4-mediated blast resistance and OsPAO4 and conversely, overexpressing OsPAO4 enhanced resistance to M. oryzae (Figure 6A,B) and upregulated PR genes (PR1a, PR1b, and PR10a) (Figure 6A–E). The ko-ospao4 lines exhibited lower levels of H₂O₂ compared to the wild-type (Figure 5E), while the overexpression lines displayed elevated levels of H₂O₂ compared to the wild-type (Figure 6F). Additionally, the activities of key ROS-scavenging enzymes, such as SOD, POD, and CAT, were significantly higher in the ko-ospao4 lines (Figure 5F–H) while low in overexpression lines (Figure 6G–I). This suggests that the capacity to scavenge ROS in the knockout lines and overexpressing lines are contrary, which may contribute to the susceptibility and resistance against M. oryzae in the two lines, respectively. These findings collectively suggest that the absence of OsPAO4 leads to an altered redox balance, with the ko-ospao4 lines displaying a greater capacity to scavenge ROS. Importantly, after inoculation with M. oryzae, H₂O₂ levels in the ko-ospao4 lines were notably reduced compared to the wild type (Figure 5I). This reduction in H₂O₂ accumulation in response to pathogen attack further underscores the compromised resistance in the absence of OsPAO4.

Our findings collectively underscore the important role of OsPAO4 in rice blast resistance. The observed alterations in ROS dynamics in the absence or overexpression of OsPAO4 suggest a potential link among PAs, ROS signaling, and plant immunity. PAs have been implicated in various stress responses in plants, and their crosstalk with ROS signaling pathways merits further investigation [13,18,22]. Additionally, the influence of OsPAO4 on immunity suggests that it may be a key player in the early stages of plant defense responses, and these results also indicate that R gene-mediated ETI depends on the OsPAO4-mediated pathway. In conclusion, our study elucidates the multifaceted role of OsPAO4 in modulating rice immunity against M. oryzae.

3.4. Association of OsPAO4 with Polyamine Metabolism and Ethylene Pathway

PA catabolism serves as a significant pathway for the production of H₂O₂ [49]. This process involves polyamine oxidases (PAOs), which generate H₂O₂ through the oxidative breakdown of the Put, Spd, and Spm. Among the seven PAOs, OsPAO1, OsPAO3, OsPAO4, and OsPAO5 are responsible for catalyzing PA back-conversion reactions of PAs, including Spd, Spm, and Put [44]. This oxidation by PAOs results in the formation of the respective amino aldehydes, 1,3-diaminopropane, and H₂O₂ [50]. Given OsPAO4’s possible involvement in controlling H₂O₂ levels and its impact on resistance against rice blast disease, we explored the role of OsPAO4 on PAs levels. Remarkably, both knockout and overexpression of OsPAO4 resulted in significant accumulation of Spd and Spm (Figure 7A,B), while Put levels did not significantly change in both OsPAO4 overexpressing and OsPAO4 knockout plants (Figure 7C). This observation suggests that OsPAO4 plays a pivotal role in modulating the levels of Spd and Spm, with implications for rice blast resistance. Further investigations post-inoculation with the rice blast pathogen unveiled intriguing dynamics in PA levels. In OsPAO4 knockout mutants, Spd rapidly degraded post-inoculation but then stabilized at 12 and 24 hpi (Figure 7B). Conversely, Spm levels consistently decreased (Figure 7A). These fluctuations in polyamine levels correlated with altered disease susceptibility, reinforcing the importance of Spm in maintaining rice blast disease resistance (Figure 7A). On the other hand, overexpression of OsPAO4 led to a transient decrease in Spd and Spm levels, followed by an increase after pathogen inoculation (Figure 7A,B). This dynamic response underscores the significance of these PAs in the rice immune response against M. oryzae. The connection between OsPAO4 and polyamine metabolism is of great interest due to the potential impact on rice’s defense strategies. PAs are known to participate in various cellular processes, including stress responses. Previous research has demonstrated that Spm can function as a plant defense activator, enhancing the plant’s resistance to stress [51]. In fact, our study has shown that both Spm and Spd can enhance resistance to rice blast disease, but Spm’s enhancement is more significant (our unpublished data). Our findings suggest that OsPAO4 is a central player in regulating polyamine levels, contributing to rice’s ability to combat M. oryzae infection.

Notably, PAs and ethylene share a common precursor, S-adenosyl-L-methionine (SAM), in their biosynthesis pathways [52]. Alterations in polyamine levels could thus influence ethylene synthesis and metabolism, subsequently modulating the plant’s immune response. Our results revealed that knockout of OsPAO4 resulted in an upregulation of the transcriptional repressor OsERF3, accompanied by a decrease in ethylene production [53]. This shift in ethylene dynamics was associated with increased susceptibility to M. oryzae. Furthermore, this trend corresponded with the downregulation of key ethylene synthesis-related genes, including OsACO2, OsACO3, and OsACS1 (Figure 7D).

These findings suggest that OsPAO4 plays a crucial role in regulating rice’s resistance to M. oryzae by controlling PA accumulation and modulating the ETH pathway. The crosstalk between PAs and ethylene, both of which are pivotal in plant stress responses, provides a compelling avenue for further research into the intricate network of signaling pathways involved in rice immunity.

4. Methods

4.1. Plant Materials and Growth Conditions

Rice (Oryza sativa L.) plants used in this study were the blast-susceptible japonica accession Lijiangxintuanheigu (LTH) and the blast-resistant accession Pik-H4 NIL (including disease resistance gene Pik-H4) [54]. All these rice plants were grown in a growth chamber at a temperature of 28 °C and 70% relative humidity with 14 h light, followed by 10 h dark, while the N. benthamiana plants, used for Agrobacterium-infiltration experiments, were maintained in a growth chamber at a temperature of 22 °C and 14 h light, followed by 10 h dark.

4.2. Plasmid Construction and Genetic Transformation

To create the Osa-miR11117 overexpression (OX11117), Osa-miR11117 target mimicry (MIM11117), and OsPAO4 overexpression (OX11117) plasmid constructs, pOX plasmid was utilized [55]. Osa-miR11117 precursor sequence, target mimicry sequence, and coding sequence of OsPAO4 were cloned into BamHI–HindIII; sites of the binary vector pOX plasmid, respectively. For the CRISPR/Cas9 plasmids of KO11117 and ko-ospao4, target primers (Table S2) of Osa-miR11117 and OsPAO4 were designed respectively according to the CRISPR/Cas9 online target design website (http://crispr.hzau.edu.cn/CRISPR2/ accessed on 20 August 2019). Then, using the pGRT vector as a template, PCR amplification was performed to obtain the gRNA-target sequence-tRNA module, which was then cloned into the Cas9 protein expression vector pRGEB32, respectively. After colony PCR and subsequent sequencing verification of the above constructs, the positive transformants were transformed into rice blast-resistant accession Pik-H4 NIL through Agrobacterium-mediated methods, respectively.

For the reporter plasmids, we introduced artificial sequences (OsPAO4ts and OsPAO4mts) of the Osa-miR11117 target gene site at the beginning of the eGFP codon sequences. These constructs and the Osa-miR11117 precursor sequence were then inserted into the KpnI site of the binary vector pEXT06G1, respectively. These constructed plasmids were subsequently employed in Agrobacterium-mediated transient expression assays in N. benthamiana plants.

4.3. Pathogen Growth, Infection, and PAs Treatment

In this study, we utilized two strains of M. oryzae, namely Guy11 and GDYJ7. To cultivate these M. oryzae strains, we used a complete medium (CM) as described by Chandran et al. [29]. These strains were cultured at 28 °C under a 12 h light/12 h dark cycle to induce sporulation. After two weeks of cultivation, spores were collected, and their concentration was adjusted to 5 × 10⁵ spores mL⁻¹ for the subsequent assays. For spray inoculation, four-leaf-stage seedlings were inoculated with the spores. The disease phenotypes on the second leaf were recorded at 6 dpi. In the punch inoculation method [56], 5 µL of the spore suspension was applied at two spots on each leaf, and the leaves were kept in a culture dish containing 0.1% 6-Benzylaminopurine (6-BA). Lesion length was measured after 6 dpi. We calculated the relative fungal mass by comparing the DNA concentration of M. oryzae Pot2 against the rice genomic ubiquitin DNA level using qPCR, following the protocols by Li et al. [27].

For PAs treatment, different concentrations of Spd (10 mM), Spm (10 mM), and Put (10 mM) were sprayed on four-leaf-stage seedlings, and samples were collected at indicated time points, respectively. Similar results were obtained from three independent experiments.

4.4. GUS Staining Assay

An approximately 2 kb promoter sequence of Osa-miR11117 precursor was cloned and inserted into the pCAMBIA1305 vector. The resulting P_Osa-miR11117-GUS plasmid was then introduced into Agrobacterium tumefaciens strain EHA105 for infection of the wild-type Pik-H4 NIL callus to obtain the P_Osa-miR11117: GUS transgenic rice plants. Subsequently, the transgenic plant tissues were subjected to a series of washes using a 100 mM Na₃PO₄ buffer (pH 7.0) three times. Following the washes, the tissues were incubated with a GUS-staining solution. This solution contained 100 mM Na₃PO₄ (pH 7.0), 10 mM EDTA, 2 mM 5-bromo-4-chloro-3-indolyl-β-GlcA, 5 mM K₄Fe(CN)₆, 5 mM K₃Fe(CN)₆, and 0.2% Triton X-100. The incubation was carried out at a temperature of 37 °C, ranging from 20 min to 24 h, as per the method described by Jefferson et al. [57]. Similar results were obtained from three independent experiments.

4.5. RNA Isolation, Quantitative RT-PCR, and Northern Blot

To isolate total RNA from collected samples, we used TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription to generate cDNA was performed using the SuperScript first-strand synthesis kit (Invitrogen). The resulting cDNA was used for the qPCR reactions, which were performed using SYBR Green Real-Time PCR Master Mix (Toyobo, Osaka, Japan), following the manufacturer’s instructions. For miRNA expression analysis, we employed the stem-loop pulse qRT-PCR method [58], utilizing designed stem-loop primers for reverse transcription and forward primers containing the 5′ end of mature miRNA sequences (both forward and reverse primers) (Table S2) based on the design by Varkonyi-Gasic et al. [58]. For internal reference, U6 snRNA-specific primers were used. To normalize the expression of target genes, the rice ubiquitin (UBQ) gene served as the internal reference. Relative expression of the target genes was calculated using the 2^–ΔΔCT method. Each experiment was carried out with three biological repeats [54].

An amount of 3 μg of total RNA was separated on a 12% denaturing polyacrylamide gel and then transferred to Hybond-N+ membranes (GE, Amersham, Boston, MA, USA) at a current of 400 mA for 1 h. To crosslink the RNA, UV light was applied using the HL-2000 HybriLinker (UVP) for 2 min. Following this, the membrane was placed in Dig Easy Hyb solution (Roche, Basel, Switzerland) at 42 °C for 1 h, and subsequently, it was hybridized with the 5′-Dig labeled miRNA probe in the same hybridization buffer, with incubation at 42 °C overnight. The membrane was then washed using the Dig Wash and Block Buffer Set (Roche) as per the manufacturer’s instructions.

4.6. Agrobacterium-Mediated Transient Expression Analysis in N. benthamiana

Agrobacterium strain GV3101 containing the individual constructs was cultured at 28 °C overnight in LB media supplemented with kanamycin (50 mg/mL), rifampin (50 mg/mL), and gentamicin (50 mg/mL) in a shaker. The bacteria were then collected by centrifugation, and the pellet was resuspended in an MMA buffer (consisting of 10 mM MgCl₂, 10 mM MES, and 200 mM acetosyringone). Subsequently, the Agrobacteria containing the constructs were introduced into the leaves of N. benthamiana by infiltration using a 1 mL syringe without a needle for a transient expression assay. The fluorescence signal was observed at 48 hpi using a confocal microscope. Simultaneously, samples were collected, and a western blot assay was conducted. Briefly, 100 mg of fresh N. benthamiana leaves were used to extract total protein with a protein extraction buffer (comprising 0.25 M Tris-HCL, pH 6.8; 4% SDS; 0.1% bromophenol blue; and 40% glycerol). The total proteins were separated using 12% SDS-PAGE and transferred onto a PVDF membrane (Millipore, Darmstadt, Germany) using Trans-Blot Turbo (BIO-RAD, Hercules, CA, USA). The polyclonal anti-GFP antibody (1:2000) was used to interact with the target protein, and the Clarity™ Western ECL Substrate system (BIO-RAD) was employed to detect GFP accumulation. Similar results were obtained from three independent experiments.

4.7. H₂O₂ Measurement and Enzymatic Activity Assay

To assess H₂O₂ levels and the activities of the enzymes SOD, POD, and CAT, 100 mg of fresh leaf samples by homogenizing them with 900 µL of PBS (pH 7.4, 0.1 M), according to the ratio of weight (g):volume (mL) = 1:9, using a glass homogenizer. After centrifugation at 3500× g for 12 min, the supernatant was used for analysis. Commercially available test kits from Nanjing Jiancheng Bioengineering Institute, Nanjing, China, were employed for the assays.

H₂O₂ content was determined using the H₂O₂ Assay kit (A064-1-1), following the manufacturer’s instructions. H₂O₂ forms a complex with molybdate, which was quantified by measuring absorbance at 405 nm and expressed as mmol gprot⁻¹. SOD activity was determined using the SOD Assay kit (A001-1), following the manufacturer’s instructions. Following thorough mixing, the samples were incubated at 37 °C for 20 min, and subsequently, 200 μL of the mixture was extracted and placed in a 96-well enzyme standard plate. The absorbance was then measured at 450 nm. SOD activity was defined as one unit, corresponding to the amount of enzyme required to achieve a 50% inhibition rate of SOD, and expressed as U mgprot⁻¹. POD activity was measured using the POD Assay Kit (A084-3-1) with reagents added as per the manufacturer’s instructions. After thorough mixing, the samples were centrifuged at 3500 rpm for 10 min, and 200 μL of the resulting supernatant was transferred to a 96-well enzyme standard plate. Subsequently, the absorbance was recorded at 420 nm and expressed as U mgprot⁻¹. CAT activity was assessed using the CAT Assay Kit (A007-1), and reagents were added according to the manufacturer’s protocol. After mixing, 200 μL was added to a 96-well enzyme standard plate. The absorbance was measured at 405 nm and expressed as U mgprot⁻¹.

The soluble protein content was also determined using the protein quantitative test box (A045-4-2). Three experimental replicates and three biological replicates were performed for each sample. According to the instructions, the optical density (OD) value of each reaction mixture at 562 nm wavelength was determined by a microplate spectrophotometer, and the soluble protein content was calculated by combining the formula. In brief, H₂O₂ content (mmol gprot⁻¹) = (measured OD value − blank OD value)/(standard OD value − blank OD value) × standard substance concentration /Protein concentration of the sample to be measured. POD activity (U gprot⁻¹) = (determination OD − control OD)/(12 × cupola light diameter) × (1000 × total volume of reaction liquid (mL))/sample size (mL)/reaction time/protein concentration of the sample to be measured. SOD activity (U gprot⁻¹) = (control OD value − measured OD value)/control OD value/ 50% × total volume of reaction liquid (mL)/sample amount (mL)/protein concentration of the sample to be measured. CAT activity (U gprot⁻¹) = (determination OD − control OD) × 235.65 × 1/60 × sample size (mL)/protein concentration of the sample to be measured (265.65 is the slope derivative).

4.8. Extraction and Analysis of PAs

PAs, including Spm, Spd, and Put, were quantified using a modified method based on Zapata et al. [59]. Leaf samples weighing between 100 and 200 mg were ground into powder using liquid nitrogen and then immersed in 1 mL of 5% trichloroacetic acid (TCA) at 4 °C for 1 h. Afterward, the samples underwent centrifugation at 12,000 rpm at 4 °C for 30 min, and 500 μL of the resulting supernatant was transferred to a new 15-mL tube.

To this supernatant, 7 μL of benzoyl chloride and 1 mL of 2 mol L⁻¹ NaOH were added, followed by vortexing for 20 s. The mixture was then incubated at 37 °C for 20 min. Subsequently, 2 mL of saturated NaCl and 1.5 mL of ethyl ether were introduced, and the mixture was centrifuged at 1500× g for 10 min at 4 °C. In a new tube, 1 mL of the ether phase was carefully collected and subjected to vacuum drying. The resulting residue was dissolved in 150 μL of high-performance liquid chromatography (HPLC) grade methanol.

For HPLC analysis, an ODS-BP column (5 μm, 4.6 mm × 200 mm, Elite, Shenzhen, China) was used in a system consisting of a ddH₂O pump-A and a gradient (acetonitrile) pump-B, along with a UV detector. A total of 20 μL of samples were injected, and PAs were eluted using a mobile phase composed of acetonitrile and ddH₂O (in a 50:50 ratio, v/v) at a flow rate of 1 mL per minute. The benzoyl-PAs were detected via absorbance at 254 nm. The results are presented as μg per gram of fresh weight (μg g⁻¹ FW) and represent the mean ± SE of extractions from three distinct samples for each treatment. Each experiment was carried out with three biological repeats.

5. Conclusions

Osa-miR11117 negatively regulates blast resistance by targeting OsPAO4 to suppress its expression and maintain Spd and Spm at a relatively stable level to cope with changes in both internal and external environments. Our findings suggest that OsPAO4 may regulate rice blast resistance by influencing PA metabolism and the ethylene signaling pathway. The findings provide valuable insights into the complex interplay between polyamine metabolism, ROS signaling, and plant defense mechanisms. Further research is warranted to unravel the precise molecular mechanisms underlying OsPAO4-mediated resistance and its potential applications in enhancing rice blast resistance in agricultural settings.