Ubiquitin Ligase Gene OsPUB57 Negatively Regulates Rice Blast Resistance

Abstract

The ubiquitination and degradation of proteins are widely involved in plant biotic and abiotic stress responses. E3 ubiquitin ligases play an important role in the ubiquitination of specific proteins. In this study, we identified the function of a U-box E3 ubiquitin ligase gene OsPUB57 in rice. Expression analyses revealed that OsPUB57 was mainly expressed in the aboveground part of rice. Drought, salt, cold, JA (jasmonic acid), PAMPs (pathogen-associated molecular patterns) or Magnaporthe oryzae treatment could significantly suppress the expression of OsPUB57 in rice. Compared with wild-type plants, OsPUB57-overexpressing plants showed a decrease in resistance to M. oryzae, while the mutant plants exhibited an enhancement of M. oryzae resistance. The expression level detection indicated that OsPUB57 negatively regulates rice blast resistance, probably by down-regulating the expression of the defense-related genes OsPR1a and OsAOS2. This study provides a candidate gene for the genetic improvement of rice blast resistance.

1. Introduction

The ubiquitin-proteasome system regulates protein degradation through protein polyubiquitination and 26S proteasome in eukaryotes [1,2]. In plants, this system mediates a series of life activities by degrading specific proteins, including DNA repair, tissue differentiation, signaling transduction, adversity adaptation and immune response. The process of protein ubiquitination mainly involves three enzymes, namely ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2) and ubiquitin ligase (E3), which are indispensable for the binding process of ubiquitin (UB) to the target protein [3,4]. E3 ubiquitin ligases can specifically recognize target proteins, which is crucial to the ubiquitin-proteasome system’s ability to specifically degrade target proteins. According to the structure and ubiquitin transfer mechanism, E3 ubiquitin ligases can be divided into two major classes including HECT (homologous to E6-associated protein C-Terminus) and RING (really interesting new gene) finger/U-box. HECT proteins can form a HECT-Ub-thioester intermediate before the ubiquitination of a substrate. RING finger/U-box proteins can directly transfer ubiquitin molecule from E2-UB intermediate to the substrate [5,6]. Generally, the U-box domain is composed of 70 amino acids with the lack of cysteine and histidine residues required for zinc chelation. It belongs to the modified RING finger domain [7,8]. U-box ubiquitin ligases are widely present in eukaryotes. There are two U-box E3 proteins in yeasts [9] and 21 U-box E3 proteins in humans [10]. In plants, 64 U-box E3 ubiquitin ligase-encoding genes are predicted in Arabidopsis thaliana [11], 77 in rice [12], and 121 in moso bamboo [13]. These E3 proteins with the U-box domain in plants are usually designated as PUBs (Plant U-box proteins).

PUBs are involved in the regulation of plant growth, development and various stress responses. In rice, OsPUB2 and OsPUB3 play important roles in responses to cold stress [14]. OsPUB4 was predicted to be involved in the regulation of the diurnal rhythm [15]. OsPUB9 negatively regulates the resistance to bacterial leaf blight [16]. OsSPL11/OsPUB13 and OsCIE1/OsPUB12 negatively regulate plant immunity through the ubiquitination of target proteins [17,18]. OsPUB15 influences cell death and cellular reactive oxygen species (ROS) levels [12,19]. The overexpression of OsPUB23 in rice resulted in higher plant yield due to an increase in grain size and weight [20]. OsPUB24 ubiquitinates the target protein OsBZR1 and thereby becomes a negative regulator in the BR signaling pathway [21]. OsPUB33 controls rice grain size and weight by regulating the OsNAC120-BG1 module [22]. OsPUB41 and OsPUB7 play a negative role in drought stress tolerance [23,24], while OsPUB67 plays a positive role [25]. OsPUB43 affects rice grain size and weight by modulating the transcription level of BR-responsive genes and MADS-box genes [26]. OsPUB44 positively regulates immune response and bacterial blight resistance [6]. OsPUB73 is involved in rice pollen development and disease resistance [27,28]. OsPUB75 (TUD1) affects rice growth and development and also negatively regulates drought tolerance [29,30].

The 77 PUB genes in rice can be classified into eight subfamilies based on their protein domains, with subfamily IV comprising 16 proteins that contain both the kinase domain and the U-box domain [12]. The functions of the proteins in this subfamily remain unexplored. OsPUB57(Os03g0424200) encodes a PUB of subfamily IV in rice. Previous studies have shown that the expression of OsPUB57 was markedly influenced by M. oryzae, the fungus responsible for rice blast [12]. In this study, the expression profiling of OsPUB57 was examined under various biotic and abiotic stress treatments to assess its potential involvement in rice defense responses. Transgenic rice lines of OsPUB57 were generated and subjected to phenotypic analyses of their rice blast resistance. The results indicate that OsPUB57 negatively regulates resistance to rice blast. Gene expression detection results revealed that OsPUB57 may suppress rice blast resistance by regulating the expression of the disease resistance-related genes OsPR1a and OsAOS2. In conclusion, OsPUB57 shows a potential as a candidate gene for future breeding and varietal improvement aimed at enhancing resistance to rice blast.

2. Results

2.1. Molecular Characteristics of OsPUB57 and Its Promoter

OsPUB57 encodes a U-box E3 ubiquitin ligase that consists of 11 exons and 10 introns. Its full-length cDNA is 3633 bp, including 1769 bp 5′-UTR, 307 bp 3′-UTR and a 1557 bp coding region (Figure S1). OsPUB57 encodes 518 amino acids with a kinase domain and a U-box domain (Figure S1). After searching for homologous sequences, the eight most homologous sequences of OsPUB57 protein from different species (Triticum turgidum, Triticum dicoccoides, Triticum aestivum, Triticum Urartu, Hordeum vulgare, Brachypodium distachyon, Sorghum bicolor, and Miscanthus lutarioriparius) were selected for sequence alignment and phylogenetic tree analysis. The results shown in Figure 1A show that all nine sequences have the kinase domain and U-box domains, and the proportion of conserved amino acids in the nine sequences reaches 76.8%. The phylogenetic tree in Figure 1B reveals that OsPUB57 occupies a distinct branch. The homologous sequences from wheat, barley and B. distachyon were clustered into a branch. The homologous sequences from S. bicolor and M. lutarioriparius belong to a branch. In addition, when searching for the homologous sequences, no sequences from dicotyledonous plants were found in the top 100 homologous sequences.

Bioinformatics analyses revealed that OsPUB57 has many cis-acting elements in its promoter region (Supplemental Table S2), such as a fungal elicitor response element (Box-W1), two JA signal transduction elements (CGTCA-motif and TGACG-motif), one heat stress response element (HSE), two low-temperature response elements (LTR), one drought stress response element (MES), two defense and stress response elements (TC-rich repeats), five light response elements (G-box, GT1-motif, MNF1 and Sp1(2)), three endosperm regulatory elements (GCN4-motif and Skn1-motif(2)) and so on.

2.2. Expression Profile of OsPUB57

The detection results of transcription levels demonstrated that OsPUB57 was more highly expressed in rice leaves or leaf sheaths than in roots at the seedling stage (Figure 2A). At the heading stage, the expression level of OsPUB57 was the highest in the leaf, followed by the stem, leaf sheath, panicle, and lowest in the root.

The expression profile of OsPUB57 in response to abiotic stresses showed that the expression level of OsPUB57 in rice seedlings decreased rapidly after salt treatment and was only about 20% after 12 h of treatment relative to that before treatment (Figure 2B). After cold treatment, the expression level of OsPUB57 gradually decreased and reached its lowest after 12 h of treatment (Figure 2C). Under drought treatment, its expression showed no significant change within the first 3 h, began to decrease significantly after 6 h of treatment and reached the lowest level at 12 h of treatment (Figure 2D).

Under MeJA (methyl jasmonate) treatment, the expression of OsPUB57 gradually decreased and reached its lowest level at 12 h of treatment, which was only about 20% of that before treatment (Figure 3A). Under SA (salicylic acid) treatment, the expression level of OsPUB57 first decreased and then increased, and was the lowest at 3 h of treatment, which was about 65% of that before treatment (Figure 3B).

The expression profile of OsPUB57 in rice seedlings in response to M. oryzae inoculation revealed that its expression level decreased rapidly in the first 24 h after inoculation, followed by a further decrease at 48 h, and remained at a low level until 96 h (Figure 4A), indicating a significant response of OsPUB57 to M. oryzae infection. When pathogens invade plants, the PTI (PAMP-triggered immunity) pathway in plants will be triggered by recognition of the PAMPs (pathogen-associated molecular patterns) of the pathogen, which in turn induces a series of signal transduction processes and changes in the expression of immunity-related genes. The expression level of OsPUB57 decreased by more than 40% at 1 h, and by about 80% at 6 h after flg22 (PAMP) treatment of the rice seedlings. Under chitin (PAMP) treatment, the expression level of OsPUB57 in rice seedlings rapidly decreased to 20% of that before treatment at 1 h of treatment, followed by slow increases, and was about 60% of that before treatment at 6 h (Figure 4B). These results indicated that OsPUB57 is very likely involved in rice’s innate immune response.

2.3. OsPUB57-Overexpressing Plants Showed Decreased Rice Blast Resistance

OsPUB57-overexpressing rice plants were created by Agrobacterium-mediated transgenic technology, and three overexpression lines (OX-11, OX-12 and OX-17) derived from different individual plants of T₀ generation were obtained. Expression level detection procedures revealed that the OX-11, OX-12 and OX-17 lines had significantly higher expression levels of OsPUB57 than the wild-type plants (WT, Nipponbare) (Figure 5A). These three overexpression lines were further inoculated with M. oryzae for blast resistance identification. As a result, the plants of the OX-11, OX-12 and OX-17 lines showed a more susceptible phenotype than the WT plants (Figure 5B). The overexpression lines had about twofold leaf lesion areas relative to the WT (Figure 5C), as well as significantly higher relative fungal growth (Figure 5D). These results indicated that OsPUB57-overexpressing plants had significantly less rice blast resistance than the WT plants.

2.4. The Mutant Plants of OsPUB57 Displayed Improved Rice Blast Resistance

A target sequence (Supplemental Table S3) was selected from the DNA sequence of OsPUB57 for gene-editing, and the corresponding transgenic rice plants were created. After PCR, sequencing and seed reproduction, three homozygous frameshift mutants, including M-1, M-22 and M-35, were selected as the mutation materials for OsPUB57 (Supplemental Table S3). The frameshift mutations in the OsPUB57 gene of the three mutants all led to premature termination of its translation, resulting in the loss of function of the OsPUB57 protein (Figure S2). The evaluation results of rice blast resistance (Figure 6A) showed that the mutant plants had a significantly lower disease degree than the wild-type plants (WT, Nipponbare). Figure 6B shows that the leaf lesion area of the mutants was about 20–30% that of the WT. Figure 6C reveals that the relative growth of M. oryzae in the leaves of mutant plants was only about 10–20% that of the WT leaves.

2.5. OsPUB57 May Influence the Expression of Defense-Related Genes to Regulate Rice Blast Resistance

To further investigate the mechanism by which OsPUB57 regulates rice blast resistance, the expression levels of two defense-related genes, OsAOS2 (allene oxide synthase 2) and OsPR1a (pathogenesis-related 1a) [31,32,33], were measured in the mutant plants of OsPUB57 before and after inoculation with M. oryzae. The results showed that, prior to inoculation with M. oryzae (0 h), the expression levels of OsPR1a in mutants M-1 and M-22 were significantly higher than those in the wild type (WT), while the expression level of OsAOS2 in the mutants was significantly lower than that in the WT. However, 24 h after inoculation with M. oryzae, the expression level of both genes in the mutants was significantly higher than those in the WT. (Figure 7A,B).

3. Discussion

Temporal and spatial expression analyses revealed that the expression level of OsPUB57 was the highest in leaves and extremely low in roots at both the seedling and heading stages, suggesting that OsPUB57 should function in the aerial part but not in the underground part of rice in its natural environment. In addition, OsPUB57 was responsive to some abiotic stress treatments. Its expression level changed greatly under salt treatment, indicating that it may be involved in regulating the salt tolerance of rice.

Usually, the innate immune response of rice will be activated upon inoculation with M. oryzae, through a series of signaling transduction processes named as PTI (PAMP-triggered immunity) or ETI (effector-triggered immunity), and the expression of defense-related genes will be induced for defense against pathogens [34,35]. The down-regulation of OsPUB57 in response to PAMP (chitin and flg22) treatments or M. oryzae inoculation indicates that OsPUB57 may be a negative regulator in the PTI pathway or in both the PTI and ETI pathways of rice. This speculation was also confirmed by the results of M. oryzae inoculation on OsPUB57-overexpressing plants and mutant plants. Disease resistance phenotype and statistics results of lesion areas or fungal growth revealed that OsPUB57 negatively regulates rice blast resistance, and its mutation can improve rice blast resistance. Downstream of ETI or PTI, the activation of complex phytohormone signaling networks is crucial for stimulating the plant’s immune signaling network. SA and JA play key roles in plant signaling networks involved in local or systemic defense responses against multiple pathogens [36,37,38]. Either JA or SA treatment would induce the expression of the pathogenesis-related (PR) gene OsPR1 in rice. Previous research has confirmed that the active form of the NPR1 (non-expressor of pathogenesis-related genes 1) protein (SA receptor) interacts with the TGACG-binding factor (TGA) transcription factor family protein, which could bind to the cis-elements of the PR1 promoter to regulate the expression of PR1 [39]. OsPR1 serves as an important marker for systemic acquired resistance (SAR) [33]. In this study, both before and after inoculation with M. oryzae, the expression level of OsPR1a in the mutant plants of OsPUB57 was significantly higher than that in the wild-type plants. This played a positive role in enhancing the disease resistance of the mutant plants. OsAOS2, a key gene in the JA synthesis pathway [31,40], showed significantly lower expression levels in the mutant plants before M. oryzae inoculation, while its expression level in the mutant plants was significantly higher than that in the wild-type plants after inoculation. OsAOS2 was strongly induced in the mutant plants after M. oryzae inoculation. These results suggest that in the absence of M. oryzae infection, the mutation of OsPUB57 in rice may lead to a decrease in JA synthesis. However, once the rice plants with mutated OsPUB57 are infected with M. oryzae, they are able to induce JA synthesis more strongly through the activation of a certain pathway to resist the disease. The specific regulatory mechanism underlying this response needs further research. Previous studies have found that after being inoculated with M. oryzae, the expression level of OsPUB57 only increased in the rice plants carrying the Pi9-resistant gene, while it decreased in rice plants without Pi9 [12]. This is consistent with the results on Nipponbare (NPB) in this study, indicating that OsPUB57 may play different roles in different immune pathways of PTI and ETI in rice.

OsPUB57 was previously reported to have E3 ubiquitin ligase activity [12]. E3 ubiquitin ligase usually performs its biological function by mediating the ubiquitination of a specific substrate. Some PUBs in rice have been revealed to have biological functions, and some specific ubiquitination substrates have been identified. Among the 77 PUBs of rice, OsPUB9, OsCIE1(OsCERK1-interacting-E3 1)/OsPUB12, OsPUB73, OsPUB15, OsPUB44 and SPL11 (SQUAMOSA promoter binding protein-like 11)/PUB13 have been found to be involved in rice’s innate immune system. OsPUB73 positively regulates rice resistance against M. oryzae and Xoo by interacting with OsVQ25 (valine-glutamine (VQ) motif-containing protein 25) and promoting its degradation via the ubiquitin-26S proteasome pathway. OsVQ25 plays an important role in balancing rice immunity and growth through interaction with OsPUB73 and a transcription factor OsWRKY53 (tryptophan (W)-arginine (R)-lysine (K)-tyrosine (Y) 53) [28]. Rice U-box E3 ubiquitin ligase SPL11 ubiquitinated and degraded a Rho GTPase-activating protein SPIN6 (SPL11-interacting protein 6) to regulate the activity of small GTPase OsRac1 (Ras-related C3 botulinum toxin substrate 1) in rice cells, which could transfer the defense signals from SPL11 to OsRac1 to regulate plant cell death (PCD) and innate immunity [17]. OsPUB15 directly interacted with the kinase domain of PID2K and regulated PCD and blast disease resistance. PID2K can phosphorylate OsPUB15, and only the phosphorylated form of OsPUB15 has E3 ligase activity [2]. When pathogens infect plants, their effector proteins are delivered to host cells to suppress plant immunity. It is interesting that OsPUB44 is the target of an effector (XopP_Xoo) of the rice pathogen Xanthomonas oryzae pv. oryzae. XopP_Xoo directly interacts with the U-box domain of OsPUB44 and inhibits its ligase activity to suppress PGN- and chitin-triggered immunity (PTIs) and disease resistance [6]. OsCIE1/OsPUB12 ubiquitinates and curtails the kinase activity of OsCERK1 (chitin elicitor receptor kinase 1) at a basal level during homeostasis, serving as a molecular brake that prevents OsCERK1-mediated autoimmunity. It is unusual that OsCIE1-mediated ubiquitination of OsCERK1 does not mark OsCERK1 for proteasomal degradation but instead suppresses its kinase activity. In the presence of pathogens, this ubiquitination is blocked, which releases the brake and enables the full activation of OsCERK1-mediated immune signaling cascades that are required to protect plant cells from infection [18]. Determination of the ubiquitination target and the upstream regulator of OsPUB57 will be the key to revealing its specific regulatory mechanism in rice immunity.

Some proteins have been predicted to be potential interactors with OsPUB57 in the STRING database (https://cn.string-db.org/ (accessed on 10 November 2024)). Function annotation results indicated that these proteins included NB-ARC domain containing protein (Os01g0314700), protein kinases (Os02g0281000 and Os01g0872800), E3 ligase (Os10g0466300), 1-acyl-sn-glycerol-3-phosphate acyltransferase (Os10g0497100), phospholipid/glycerol acyltransferase (Os04g0625200) and so on. NB-ARC proteins are well-known for their involvement in disease resistance [41]. An activated form of the NB-ARC protein RLS1 functions with cysteine-rich receptor-like protein RMC to modulate the oxidative state, cell death process, and associated immunity responses in rice [42]. Protein kinases have been shown in multiple studies to play crucial roles in regulating rice disease resistance. Overexpression of the calcium-dependent protein kinase OsCPK4 in rice enhanced the resistance to M. oryzae [43]. The protein kinase OsCIPK31 (CBL-interacting protein kinase 31), in conjunction with OsCBL2 (calcineurin B-like 2) and OsAKT1L (AKT1-like), forms the CBL2-CIPK31-AKT1L signaling pathway, collectively regulating rice blast resistance [44]. The predicted interacting protein encoded by Os01g0872800 is the protein kinase OsPdk2, which was previously reported to regulate basal disease resistance through the OsOxi1 (oxidative signal-inducible 1)-OsPti1a phosphorylation cascade in rice [45]. Further research is needed to determine whether OsPUB57 regulates rice blast resistance by interacting with some of these proteins.

4. Materials and Methods

4.1. Materials and Growth Conditions

The japonica rice variety Nipponbare (NPB) was used for various treatments, expression level detection and genetic transformation in this study. Dehulled rice seeds were sequentially grown on 1/2 MS medium for seven days and in Hoagland nutrient solution for three weeks under controlled photoperiodic conditions (14 h light, 28 °C/10 h dark, 25 °C), and then the obtained seedlings were used for various treatments, M. oryzae inoculation, and the expression level detection of OsPUB57 in different tissues at the seedling stage. In addition, rice seedlings cultured under the same conditions were transplanted to a field to determine the expression level of OsPUB57 in different tissues at the heading stage.

4.2. RNA Extraction and qRT-PCR Amplification

The total RNA of rice tissues was extracted using the TRIzol reagent (Carlsbad, CA, USA, Invitrogen, 15596026). First-strand cDNA was synthesized via a reverse transcription reaction of 2 µg of total RNA with a reverse transcription kit (Waltham, MA, USA, Thermo Scientific, K1682). The amplification reagent used in qRT-PCR was TB Green Premix Ex Taq (Dalian, China, TaKaRa, RR420L). The total volume of the reaction system was 20 µL, and the reaction apparatus was an Applied Biosystems Stepone^TM Real-Time PCR System (Waltham, MA, USA, Thermo Fisher, ABI Step One). The reaction program of qRT-PCR was as follows: 95 °C for 1 min, followed by 40 cycles at 95 °C for 5 s, 60 °C for 30 s. The internal reference gene used for the detection of the relative expression levels of rice genes was the ubiquitin (Os03g0234200) of rice [46]. The relative expression levels of rice genes were calculated by the 2 ^−∆∆Ct method using Excel software [47,48]. A t-test was employed to assess the significance of differences using SPSS 24, and Origin 2021 Prism was utilized for plotting.

All of the primers used in this study were synthesized by Sangon Biotech (Shanghai) Co., Ltd. (Shanghai, China). The sequences and functions of the primers are presented in Supplemental Table S1.

4.3. Vector Construction and Genetic Transformation of Rice

The full-length CDS of OsPUB57 was amplified from rice whole cDNA samples. An overexpression vector of OsPUB57 (pCXUN-OsPUB57) was constructed using the zero-background T-Vector system [49]. A 20-bp fragment was selected from the genome sequence of OsPUB57 as the target sequence for gene-editing, and the corresponding adapter primers were designed to construct the CRISPR/Cas9 gene-editing vector [50]. The overexpression vector and gene-editing vector were genetically transformed into rice Nipponbare by Agrobacterium-mediated transgenic technology to obtain the overexpression and gene-editing plants of OsPUB57. The genetic transformation process was completed at Wuhan Towin Biotechnology Company Limited (Wuhan, Hubei, China).

4.4. Cultivation of Rice Blast Fungus

M. oryzae race RO1-1 was grown on oatmeal medium and cultivated in the dark at 25 °C for 1 to 2 days. When the fungi were germinated, the medium plates were moved to 25 °C for continuous light cultivation until the plates were covered with mycelia, then irradiated with blue-black light for 1 to 2 days to induce conidia. Finally, the conidia were collected.

4.5. Inoculation of Rice Blast Fungus

The conidial suspension (0.05% tween-20) of M. oryzae was prepared at a concentration of 5 × 10⁵ conidia ml-1. Freshly isolated leaves from individual rice lines were randomly selected, punched with two holes per leaf, and then placed in a petri dish containing moist filter paper wetted with 0.1% 6-benzylaminopurine (6-BA). About 5 µL of conidial suspension was added to each hole in the leaves, and then the petri dish was sealed and moved into an incubator with high humidity at 25 °C, followed by cultivation for one day in the dark. After that, the photoperiod was changed to 14 h light/10 h dark, which continued for 5–7 days. Finally, the blast resistance of the rice materials was evaluated. To determine the relative lesion area, the lesion area of each sample was first measured using ImageJ (v1.8.0.345). The lesion area of the wild-type material (WT), denoted as M, was set to 1. The relative lesion area of each sample was then calculated using the formula M0/M, where M0 represents the lesion area of each individual sample. Based on previous literature [51], to detect the relative fungal growth, DNA was extracted from the infected tissues of each sample, treated with RNase A to remove RNA, and then subjected to a DNA-based qPCR assay. The threshold cycle value (C_T) of M. oryzae Pot2 DNA and the C_T of rice genomic ubiquitin DNA was measured, and the C_T of Ubq was subtracted from the C_T of Pot2. Relative fungal growth was then calculated as a ratio (Mo-Pot2/Os-Ubq) represented by the equation E^{CT(Os-UBQ) − CT(Mo-Pot2)}, in which the amplification efficiency, E, is 2 for the primer pairs designed for the respective genes. All inoculation experiments were repeated more than three times.

4.6. Various Treatments and Samplings

Rice seedlings were planted with a Hoagland nutrient solution containing 100 mM NaCl for salt stress treatment at 28 °C under light conditions [52]. Cold treatment was performed with seedlings grown in a Hoagland nutrient solution at 4 °C under light conditions [53]. Drought stress treatment was performed on rice seedlings according to the previously reported method [54]. Seedlings grown in a Hoagland nutrient solution under normal conditions were used as the control. Rice seedlings were transferred to Hoagland nutrient solutions containing 100 µM MeJA or 100 µM SA, respectively, for hormone treatments at 28 °C under light conditions [55,56]. Rice seedlings grown in a Hoagland nutrient solution without hormone were used as the control. For all of these treatments, rice samples with stems and leaves were collected at 0, 1, 3, 6 and 12 h of treatment, respectively.

For the M. oryzae treatment, the conidial suspension (0.05% tween-20) of M. oryzae was prepared at a concentration of 5 × 10⁵ conidia mL⁻¹ and sprayed on rice seedlings. Then, the rice seedlings were treated in the dark for 24 h at 25 °C under high humidity. After that, the rice seedlings were put into an incubator at high humidity under controlled photoperiodic conditions (14 h light, 28 °C/10 h dark, 25 °C). Rice samples with stems and leaves were collected at 0, 24, 48, 72 and 96 h after the conidia spray.

The rice seedlings were planted with a Hoagland nutrient solution containing 100 ng/L flg22 or 8 ng/L chitin for PAMP treatment. Rice seedlings grown in a Hoagland nutrient solution without flg22 or chitin were used as the control. Rice samples with stems and leaves were collected at 0, 1, 3 and 6 h of treatment.

RNA was extracted from every sample above, and the relative expression level of OsPUB57 was detected after reverse transcription. Significant differences were analyzed by comparing the control and treatment samples at each time point. The effectiveness of various treatments was verified by detecting the expression levels of the reported responsive marker genes through qRT-PCR. These genes included OsTSD2 (tumorous shoot development2) for drought and salt treatments [57], OsDREB1A (dehydration-responsive element binding factor 1A) for cold treatment [58], OsWrky45 (tryptophan (W)-arginine (R)-lysine (K)-tyrosine (Y) 45) for SA treatment [59], OsJAmyb (a JA-inducible MYB transcription factor) for JA treatment [59], OsPAL1 (phenylalanine ammonia lyase 1) for PAMP treatments [60] and OsHLP1 (HVA22-like protein 1) for rice blast fungus treatment [61]. The detection results of the expression levels of these marker genes indicated that the treatments successfully induced the relevant stress responses in this study (Figures S3–S8).

4.7. Bioinformatics Analysis

The homologous sequences of OsPUB57 were searched using blastP in the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 28 November 2024)), with the search parameters set to the defaults. The sequences with the highest homology in eight different species were selected from the top 50 homologous sequences for sequence alignment and phylogenetic tree analysis. Phylogenetic tree analysis was conducted by the neighbor-joining method of the MEGA (v11) software (bootstrap replicates were 1000). Multiple alignment of amino acid sequences was performed using the DNAman (v8) software. The 2000 bp genome sequence upstream of the start codon (ATG) of OsPUB57 was regarded as its promoter sequence, and the cis-acting element analysis of the promoter was performed in the PlantCARE database.

5. Conclusions

OsPUB57 is a U-Box E3 ubiquitin ligase gene. It is mainly expressed in the aboveground part of rice. Drought, salt, cold, MeJA, PAMPs (pathogen-associated molecular patterns) or M. oryzae treatment on significantly suppressed the expression of OsPUB57 in rice. The mutation of OsPUB57 notably enhanced rice blast resistance by upregulating the disease resistance-related genes OsPR1a and OsAOS2, while the overexpression of OsPUB57 in rice significantly reduced rice blast resistance. This indicates that OsPUB57 negatively regulates rice blast resistance. The predicted interacting proteins of OsPUB57 include some proteins that may be involved in the plant immune system. Future work should focus on verifying whether these proteins are real interacting proteins and ubiquitination substrates of OsPUB57 and whether these proteins are involved in the regulation of rice disease resistance.

Author Contributions

Conceptualization, Q.C. and W.T.; methodology, J.Z., Q.D. and Y.W.; validation, M.S. and F.G.; formal analysis, J.Z., Q.D. and Y.W.; investigation, M.S. and F.G.; resources, Z.W. and X.X.; data curation, J.Z., Q.D. and Z.W.; writing—original draft preparation, J.Z., Q.D. and Q.C.; writing—review and editing, Q.C. and W.T.; visualization, J.Z., Q.D.; supervision, Q.C., W.T. and Z.W.; funding acquisition, Q.C. and W.T. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The datasets generated during the current study are available from the corresponding authors on reasonable request.

Conflicts of Interest

The authors have no relevant financial or non-financial interests to disclose.

Footnotes

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Figures

Figure 1. Sequence analyses of OsPUB57 and its homologous sequences. (A) Sequence alignment of OsPUB57 and its homologous sequences from T. turgidum (VAH34870.1), T. dicoccoides (XP_037480658.1), T. aestivum (XP_044458441.1), T. urartu (EMS46603.1), H. vulgare (XP_044967372.1), B. distachyon (XP_010230025.2), S. bicolor (KAG0550666.1), and M. lutarioriparius (CAD6211570.1). The U-box domain (black line) and kinase domain (red line) are underlined; (B) Phylogenetic tree of OsPUB57 and its homologs.

Figure 2. Expression profile analysis of OsPUB57 in different rice tissues and under different stress treatments. (A) Expression levels of OsPUB57 in different tissues at the seedling and heading stages; (B–D) The expression profile of OsPUB57 under different treatments. Error bars are standard deviations based on three replicates. Analysis of significant differences in expression levels between the control and treatment samples was conducted at each time point (* represents p [less than] 0.05).

Figure 3. Expression profile of OsPUB57 in rice under different phytohormone treatments. (A) MeJA treatments and (B) SA treatments. Error bars are standard deviations based on three replicates. Analyses of significant differences in expression levels between the control and treatment samples were conducted at each time point (* represents p [less than] 0.05).

Figure 4. The expression level of OsPUB57 in response to M. oryzae inoculation (A) and PAMP treatments (B). Error bars are standard deviations based on three replicates. Analyses of significant differences in expression levels between the control and treatment samples were conducted at each time point (* represents p [less than] 0.05).

Figure 5. Rice blast resistance identification of OsPUB57-overexpressing plants (OX-11, OX-12 and OX-17) and the WT plants (Nipponbare). (A) The expression level of OsPUB57; (B) Rice blast resistance phenotypes. Bar = 1 cm; (C) Relative lesion area; (D) Relative fungal growth. Error bars indicate standard deviations from three replicates. Asterisks represent significant differences compared with the WT (* represents p [less than] 0.05).

Figure 6. Rice blast resistance evaluation of the mutant plants (M-1, M-22 and M-35) of OsPUB57 and the WT plants (Nipponbare). (A) Rice blast resistance phenotypes. Bar  = 1 cm; (B) Relative lesion area; (C) Relative fungal growth. Error bars indicate standard deviations from three replicates. Asterisks represent significant differences compared with the WT (* represents p [less than] 0.05).

Figure 7. The expression levels of two defense-related genes (OsAOS2 and OsPR1a) in the mutant plants of OsPUB57 before and after inoculation with M. oryzae. (A) The expression level of OsAOS2. (B) The expression level of OsPR1a. Error bars indicate standard deviations from three replicates. Asterisks represent significant differences compared with the WT at each time point (* represent p [less than] 0.05).

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants14050758/s1, Table S1. Primer sequences used in this study. Table S2. The cis-acting elements on the promoter region of OsPUB57. Table S3. The genotypes of target sites in three mutant lines of OsPUB57. The blue colored nucleotides indicate the target sequence of wild type. The red colored nucleotides indicate the protospacer adjacent motif (PAM). The dotted lines are the base deletions. A bold base represents the inserted base. Figure S1. Schematic structure of OsPUB57. Figure S2. The amino acid sequence comparison of OsPUB57 in Nipponbare (NPB) and three mutants (M-1, M-22 and M-35). Figure S3. The transcriptional expression level of OsTSD2 in rice under salt and drought treatments. Note: Asterisks represent significant differences compared with the control. * represents p < 0.05, ** represents p < 0.01. The same below. Figure S4. The transcriptional expression level of OsDREB1A in rice under cold treatment. Figure S5. The transcriptional expression level of OsWrky45 in rice under SA treatment. Figure S6. The transcriptional expression level of OsJAmyb in rice under JA treatment. Figure S7. The transcriptional expression level of OsPAL1 in rice under PAMP (flg22 or chitin) treatments. Figure S8. The transcriptional expression level of OsHLP1 in rice after M. oryzae inoculation.

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