Mechanism Analysis of OsZF8-Mediated Regulation of Rice Resistance to Sheath Blight

Transcription factors are key molecules involved in transcriptional and post-transcriptional regulation in plants and play an important regulatory role in resisting biological stress. In this study, we identified a regulatory factor, OsZF8, mediating rice response to Rhizoctonia solani (R. solani) AG1-IA infection. The expression of OsZF8 affects R. solani rice infection. OsZF8 knockout and overexpressed rice plants were constructed, and the phenotypes of mutant and wild-type (WT) plants showed that OsZF8 negatively regulated rice resistance to rice sheath blight. However, it was speculated that OsZF8 plays a regulatory role at the protein level. The interacting protein PRB1 of OsZF8 was screened using the yeast two-hybrid and bimolecular fluorescence complementation test. The results showed that OsZF8 effectively inhibited PRB1-induced cell death in tobacco cells, and molecular docking results showed that PRB1 had a strong binding effect with OsZF8. Further, the binding ability of OsZF8-PRB1 to ergosterol was significantly reduced when compared with the PRB1 protein. These findings provide new insights into elucidating the mechanism of rice resistance to rice sheath blight.


Introduction
Rice sheath blight is a phytopathogenic fungus disease that occurs during rice production and has become the primary disease in some rice-producing regions [1].It is caused by the necrotrophic pathogen Rhizoctonia solani Kühn with a wide host range [2].Therefore, the research on rice sheath blight is limited by the abundance of pathogen groups, unique classification systems, diverse pathogenic mechanisms, complex interaction regulation, lack of effective resistant varieties, and genetic transformation technology [3].In recent years, studies on kinases, transcription factors, hormone signals, and synthetic regulatory pathways involved in the interaction between rice and Rhizoctonia solani have further revealed the evolution of pathogens, host selection strategies, and interaction regulatory mechanisms [4].
Transcription factors are key molecules in signal transduction and transcriptional regulation in plants.WRKY, MYB, ABI1/B3, NAC, and other transcription factor families regulate rice resistance to R. solani [5].Zinc finger proteins (ZFPs) are a class of transcription factors (TFS) that self-fold by binding to Zn 2+ to form short and stable finger-like structures [6].ZFPs are widely distributed in eukaryotic genomes and play important roles in physiological and biochemical processes, such as plant hormone signal transduction, DNA recognition, RNA binding, transcription regulation, growth and development, and biotic and abiotic stress [7][8][9].Mittler et al. [10] found that the mutation of Zat10 on the C2H2 zinc finger protein of Arabidopsis thaliana enhanced plant tolerance to abiotic stress.Huang et al. [11] identified stress-related zinc finger protein genes in rice and found that ZFP245 2 of 13 plays a role in rice response to salt and drought stress.These results suggest that ZFP245 might promote the tolerance of rice to cold and drought by regulating the proline level and reactive oxygen species (ROS) scavenging capacity.Luo et al. [12] found that SlZF3 regulates plant height by directly inhibiting genes in the gibberellin biosynthesis pathway.
Gene expression is a complex process that contains many steps: RNA transcription, protein translation, and protein stability.Most of the current research on gene expression regulation has focused on transcriptional regulation [13].For instance, according to a study on maize, ncRNA GARR-2 can interact with ZmUPL1; ZmUPL1 is a kind of enzyme participating in ubiquitin-mediated protein degradation.It is proposed that such interaction will influence the activity of the enzyme, which affects its ability to degrade the downstream targets [14].
The regulation of protein interaction is an advanced view in studying pathogen and host interaction [15].During the interaction between rice and pathogens, various proteins in rice interact with each other to promote immune-related pathways and regulate the resistance of rice to sheath blight.Yang [16] found that activator of SWEET2a (AOS2) is secreted by pathogens during infection and interacts with WRKY53 and GT1 in rice to form a transcription complex, activate the expression of SWEETs, and promote the transport of carbohydrate compounds from host cells to the outside so that the pathogen can obtain nutrients.Yuan [17] found that PhyB restrained the BZR1-NAC028-CAD8B pathway to regulate rice resistance to sheath blight.Kim [18] showed that tissue-specific activation of SWEET14 with DOF11 conferred rice resistance to sheath blight.Moreover, Sun [19] found that LPA1 activated PIN1a to improve rice resistance to sheath blight.
Sterols, major components of eukaryotic cell membranes, are synthesized in the endoplasmic reticulum and then transported to the cell membrane or other membrane structures through lipid transporter and vesicle transport pathways [20].In addition to being important components of the cell membrane and other organelle membranes, sterols play an important role in the integrity of the cell membrane, the activity of membrane binding enzymes, membrane fluidity, cell viability, cell material transport, and signal transduction [21].Different species contain different sterols [22]; for example, in fungi, cell membrane sterols mainly exist in the form of ergosterols.In some plants, sterols occur mainly in the form of phytosterols [23].In yeast cells, ERGosterol is synthesized using acetyl-CoA (acetyl-CoA) as a raw material and is formed by 25 Erg (ERGosterol biosynthesis) enzymes [21].Deleting the key Erg gene prevents the cell from synthesizing ergosterol, causing the cell membrane to rupture and resulting in the death of the fungus [24].Liu [25] identified the transcription factor FgSR in Fusarium graminearum and found that it is activated by the Hog1-MAPK pathway.When FgSR is highly phosphorylated by Hog1, it enters the nucleus and it regulates SWI/SNF complex-mediated chromosome remodeling, activating ergosterol synthesis, which indicates the pathogenic mechanism of ergosterol in pathogenic fungi.In the present study, a regulatory factor, OsZF8, involved in the interaction between rice and R. solani AG1-IA, was identified.The mechanism by which OsZF8 mediates the regulatory effect of PRB1 on rice sheath blight resistance was analyzed via yeast two-hybrid (Y2H) assays, instantaneous tobacco expression, and molecular docking.The results of this study provide new insights and directions into the further revealing of the immune mechanism and host interaction regulation.

Analysis of the Expression Patterns of OsZF8
Based on the expression pattern of OsZF8 during infection by R. solani, the expression of OsZF8 responded to infection with the Y-36 strain, reaching the highest level 60 h after inoculation.The expression patterns of OsZF8 in different rice tissues showed that OsZF8 was expressed in the panicle, stem, leaf, leaf sheath, and root, with the highest expression level in the root and the lowest expression level in the panicle (Figure 1).
was expressed in the panicle, stem, leaf, leaf sheath, and root, with the highest expre level in the root and the lowest expression level in the panicle (Figure 1).

Domain and Phylogenetic Analysis of the OsZF8 Protein
The results of the OsZF8 domain and phylogenetic analysis showed that OsZF a typical ZF-C2H2 structure and is characteristic of the OsZF8 transcription factor, w is the rice OsZF8 transcription factor (Figure 2).

Validation of OsZF8 Mutant Plants
Sequencing results of the knockout mutant showed that one base A was missing 107 bp after the initiation site of the OsZF8-ko12 mutant plant, and one base C was ins

Domain and Phylogenetic Analysis of the OsZF8 Protein
The results of the OsZF8 domain and phylogenetic analysis showed that OsZF8 has a typical ZF-C2H2 structure and is characteristic of the OsZF8 transcription factor, which is the rice OsZF8 transcription factor (Figure 2).

Domain and Phylogenetic Analysis of the OsZF8 Protein
The results of the OsZF8 domain and phylogenetic analysis showed that OsZF8 has a typical ZF-C2H2 structure and is characteristic of the OsZF8 transcription factor, which is the rice OsZF8 transcription factor (Figure 2).

Validation of OsZF8 Mutant Plants
Sequencing results of the knockout mutant showed that one base A was missing from 107 bp after the initiation site of the OsZF8-ko12 mutant plant, and one base C was inserted

Validation of OsZF8 Mutant Plants
Sequencing results of the knockout mutant showed that one base A was missing from 107 bp after the initiation site of the OsZF8-ko12 mutant plant, and one base C was inserted into 107BP-108BP of the OsZF8-ko29 mutant plant (Figure 3C).The OsZF8 expression levels in the obtained OsZF8-ko12, OsZF8-ko29, OsZF8 OX1, and OsZF8 OX2 mutants were analyzed.The results showed that the OsZF8 expression levels in the OsZF8-ko12 and OsZF8-ko29 mutant plants were significantly decreased (Figure 3B).OsZF8 expression was significantly increased in the OsZF8 OX1 and OsZF8 OX2 mutant plants (Figure 3A).into 107BP-108BP of the OsZF8-ko29 mutant plant (Figure 3C).The OsZF8 expression levels in the obtained OsZF8-ko12, OsZF8-ko29, OsZF8 OX1, and OsZF8 OX2 mutants were analyzed.The results showed that the OsZF8 expression levels in the OsZF8-ko12 and OsZF8-ko29 mutant plants were significantly decreased (Figure 3B).OsZF8 expression was significantly increased in the OsZF8 OX1 and OsZF8 OX2 mutant plants (Figure 3A).

Disease Resistance Identification of OsZF8 Mutants
Resistance of the OsZF8-ko12 and OsZF8 OX mutant plants and WT plants to R. solani was determined by evaluating the relative lesion area (Figure 3E) and relative biomass (Figure 3F).The results showed that the resistance of the OsZF8-ko12 mutant plants was significantly enhanced 72 h after inoculation with the Y-36 strain (Figure 3G).The relative lesion area and relative biomass of OsZF8-ko12 were significantly reduced compared to those of OsZF8 OX and WT plants.Similarly, the resistance of the OsZF8 OX mutant plants was similar to that of WT plants, suggesting that OsZF8 negatively regulates rice resistance to rice sheath blight.

Expression Analysis of Defense-Related Genes and Transcriptional Regulatory Genes
To analyze the transcriptional regulatory mechanism of OsZF8 in regulating resistance to rice sheath blight, we analyzed changes in expression of the salicylic acid-related resistance gene NPR1, the ethylene-related resistance genes EIN2 and EIL1, the brassinolide-related susceptibility genes D2 and D61, the defense response genes PBZ1 and PRb1, and the transcriptional regulatory genes GID1 and SLR1 in the mutant and wild-type plants under pathogen stress.The results showed no significant differences in the expression levels of EIN2, EIL1, PRb1, or D2 between the mutant and wild-type plants.NPR1 and SLR1 were significantly upregulated in OsZF8-ko12, while PBZ1 and D61 were significantly upregulated in OsZF8 OX, and GID1 was significantly downregulated in

Disease Resistance Identification of OsZF8 Mutants
Resistance of the OsZF8-ko12 and OsZF8 OX mutant plants and WT plants to R. solani was determined by evaluating the relative lesion area (Figure 3E) and relative biomass (Figure 3F).The results showed that the resistance of the OsZF8-ko12 mutant plants was significantly enhanced 72 h after inoculation with the Y-36 strain (Figure 3G).The relative lesion area and relative biomass of OsZF8-ko12 were significantly reduced compared to those of OsZF8 OX and WT plants.Similarly, the resistance of the OsZF8 OX mutant plants was similar to that of WT plants, suggesting that OsZF8 negatively regulates rice resistance to rice sheath blight.

Expression Analysis of Defense-Related Genes and Transcriptional Regulatory Genes
To analyze the transcriptional regulatory mechanism of OsZF8 in regulating resistance to rice sheath blight, we analyzed changes in expression of the salicylic acid-related resistance gene NPR1, the ethylene-related resistance genes EIN2 and EIL1, the brassinoliderelated susceptibility genes D2 and D61, the defense response genes PBZ1 and PRb1, and the transcriptional regulatory genes GID1 and SLR1 in the mutant and wild-type plants under pathogen stress.The results showed no significant differences in the expression levels of EIN2, EIL1, PRb1, or D2 between the mutant and wild-type plants.NPR1 and SLR1 were significantly upregulated in OsZF8-ko12, while PBZ1 and D61 were significantly upregulated in OsZF8 OX, and GID1 was significantly downregulated in OsZF8 OX.However, the difference was less than 0.5 times.Moreover, the phenotype of the OsZF8 mutant was inconsistent with the trend in the expression of defense-related genes in response to R. solani infection (Figure 4).These results suggest that OsZF8 might not regulate rice resistance to sheath blight at the transcriptional level.
OsZF8 OX.However, the difference was less than 0.5 times.Moreover, the phenotype the OsZF8 mutant was inconsistent with the trend in the expression of defense-relate genes in response to R. solani infection (Figure 4).These results suggest that OsZF8 mig not regulate rice resistance to sheath blight at the transcriptional level.

Prb1 Interacts with OsZF8
Subcellular localization information of OsZF8 was retrieved from the Cell-PLoc we site (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2,accessed on 1 January 2023), an the results showed that OsZF8 localizes to the nucleus.A total of 60 positive clones we obtained through screening and verification of the rice cDNA yeast library.Furthermor the interaction between OsZF8 and the Prb1 protein was verified by means of one-to-on yeast two-hybrid assays (Figure 5A).Moreover, the interaction between OsZF8 and Prb was verified in tobacco leaf cells by a bimolecular fluorescence complementation test.Flu orescence signals were detected in the nucleus of the co-inoculated sites of pSPYNE OsZF8 and pSPYCE-Prb1.However, no fluorescence signals were detected in the verific tion experiment (Figure 5B).Hence, OsZF8 and Prb1 interacted in the plant nucleus.

Prb1 Interacts with OsZF8
Subcellular localization information of OsZF8 was retrieved from the Cell-PLoc website (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2,accessed on 1 January 2023), and the results showed that OsZF8 localizes to the nucleus.A total of 60 positive clones were obtained through screening and verification of the rice cDNA yeast library.Furthermore, the interaction between OsZF8 and the Prb1 protein was verified by means of one-to-one yeast two-hybrid assays (Figure 5A).Moreover, the interaction between OsZF8 and Prb1 was verified in tobacco leaf cells by a bimolecular fluorescence complementation test.Fluorescence signals were detected in the nucleus of the co-inoculated sites of pSPYNE-OsZF8 and pSPYCE-Prb1.However, no fluorescence signals were detected in the verification experiment (Figure 5B).Hence, OsZF8 and Prb1 interacted in the plant nucleus.

Effect of OsZF8 on PRB1-Induced Cell Death
As shown in Figure 5C, the Prb1, Prb1 + GFP, and BAX treatments induced cell death in tobacco plants, though no obvious cell death was observed in the ZF8 and ZF8+Prb1 treatment groups.These experimental results indicate that OsZF8 can effectively inhibit Prb1-induced cell death.
The binding capacity of the Prb1 protein and the OsZF8-Prb1 protein complex for ergosterol was then analyzed.The binding capacity of the Prb1 protein for ergosterol was −10.3 kcal/mol, and ergosterol could bind to the surface cavity of the Prb1 protein.In addition, ergosterol formed hydrogen bonds with the SER-59, TRP-60, and GLY-58 sites on the Prb1 protein, indicating that ergosterol strongly binds to the Prb1 protein (Figure 7A).The binding energy of the ergosterol and OsZF8-Prb1 protein complex was −8.1 kcal/mol.Ergosterol can bind to the surface cavity of the OsZF8-Prb1 protein complex, but ergoste-

Effect of OsZF8 on PRB1-Induced Cell Death
As shown in Figure 5C, the Prb1, Prb1 + GFP, and BAX treatments induced cell death in tobacco plants, though no obvious cell death was observed in the ZF8 and ZF8+Prb1 treatment groups.These experimental results indicate that OsZF8 can effectively inhibit Prb1-induced cell death.
The binding capacity of the Prb1 protein and the OsZF8-Prb1 protein complex for ergosterol was then analyzed.The binding capacity of the Prb1 protein for ergosterol was −10.3 kcal/mol, and ergosterol could bind to the surface cavity of the Prb1 protein.In addition, ergosterol formed hydrogen bonds with the SER-59, TRP-60, and GLY-58 sites on the Prb1 protein, indicating that ergosterol strongly binds to the Prb1 protein (Figure 7A).The binding energy of the ergosterol and OsZF8-Prb1 protein complex was −8.1 kcal/mol.Ergosterol can bind to the surface cavity of the OsZF8-Prb1 protein complex, but ergosterol can only form hydrogen bonds with THR-111 sites on the OsZF8-Prb1 protein complex.Moreover, the hydrophobic interactions between ergosterol and ASN-144, PRO-159, TRP-115, GLN-146, and TRP-104 of the OsZF8-Prb1 protein complex indicate that the binding effect of ergosterol and the OsZF8-Prb1 protein complex is weak (Figure 7B).These results indicate that the binding of OsZF8 to the Prb1 protein decreases the ability of the Prb1 protein to bind to ergosterol.
rol can only form hydrogen bonds with THR-111 sites on the OsZF8-Prb1 protein complex.Moreover, the hydrophobic interactions between ergosterol and ASN-144, PRO-159, TRP-115, GLN-146, and TRP-104 of the OsZF8-Prb1 protein complex indicate that the binding effect of ergosterol and the OsZF8-Prb1 protein complex is weak (Figure 7B).These results indicate that the binding of OsZF8 to the Prb1 protein decreases the ability of the Prb1 protein to bind to ergosterol.rol can only form hydrogen bonds with THR-111 sites on the OsZF8-Prb1 protein complex.Moreover, the hydrophobic interactions between ergosterol and ASN-144, PRO-159, TRP-115, GLN-146, and TRP-104 of the OsZF8-Prb1 protein complex indicate that the binding effect of ergosterol and the OsZF8-Prb1 protein complex is weak (Figure 7B).These results indicate that the binding of OsZF8 to the Prb1 protein decreases the ability of the Prb1 protein to bind to ergosterol.

Discussion
The classification system of Rhizoctonis spp. is unique.R. solani is divided according to the number of nuclei in a single cell into multinucleate, binucleate, and uninucleate.R. solani strains are also divided into different anastomosis groups according to whether mycelial fusion occurs.Finally, the strains are divided into different subgroups according to their anastomosis frequency, culture characteristics, host species, pathogenicity, and molecular characteristics.Rice sheath blight and maize sheath blight are caused by the AG1-IA fusion group of R. solani, which can lead to severe yield loss [26,27].
Zinc finger proteins are among the largest families of transcription factors in eukaryotes.Zinc finger proteins are divided into 10 types depending on the type of zinc finger motif, C2H2, C2HC, C2HC5, C2C2, CCCH, C3HC4, C4, C4HC3, C6, and C8 [28], of which C2H2 zinc finger proteins are the largest group.Han [29] discovered the first C2H2-type zinc finger protein, EPF1, in petunia and found fourteen EPF1-related genes.Since then, C2H2 zinc finger proteins have been found in many plants, such as Arabidopsis [30], rice [9], and tomato [31], and play important roles in plant growth and development, gene expression and the response to biological and abiotic stresses [32].Chu et al. [33] conducted a genome-wide identification of ZFP genes in three species of grapevine, and examined the VvZFP family genes involved in response to methyl jasmonate (MeJA), abscisic acid (ABA), and salicylic acid (SA).Through an interaction network between TtC2H2-ZF proteins with identified C2H2-ZF TFs in A. thaliana, Faraji (2018) found that, ZFP1, ZFP2, ZFP3, ZFP10, and ZFP11 regulate many down-stream genes involved in multiple biological processes, including stress and hormone responses [34].
Previous studies showed that zinc finger proteins not only have transcriptional regulatory activities but also can interact with other proteins to regulate protein activity, thus regulating plant growth and disease resistance.Rice OsZFP can directly recognize P8 protein of SRBSDV and regulate rice resistance to SRBSDV [35].OsZFP213 interacts with OsMAPK3 to enhance the ability of scavenging ROS and synergistically regulate the salt tolerance of rice [36].
Post-translational modifications of protein such as ubiquitination, phosphorylation, glycosylation, and polymerization can affect the stability and activity of zinc finger proteins, and thus affect their biological functions.MdBBX7/MdCOL9 can be directly ubiquitinated and degraded by E3 ubiquitin ligase MIEL1, which reduces apple tolerance to drought stress [37].CCCH-type zinc finger protein OsLIC, as a substrate of OsMAPK6, can be activated by phosphorylation of OsMAPK6 to regulate rice resistance to Xoo and Xoc [38].AtZAT6 positively modulates expression levels of stress-related genes by directly binding to the promoter region of pathogen-related genes like PR1 and PR5 [39].ZAT10 and EIN3 can be phosphorylated by MPK3/6 [40], and ZAT12 is a direct target of EIN3, so that ZAT12 also may be involved in MAPK signal cascades [41].
At present, the function of rice zinc finger proteins, OsZF8, has not been reported.In this study, we identified that the expression of OsZF8 was related to R. solani infection.Further experiments speculated that OsZF8 might regulate rice resistance to R. solani not only at the transcriptional level but also through protein regulation.Zinc fingerlike transcription factors have protein-regulatory functions in addition to transcriptional regulation; therefore, the regulatory function of OsZF8 was further studied.The OsZF8interacting protein Prb1 was screened through tests in vitro and in vivo.The Prb1 protein, a downstream functional protein involved in rice defense response to disease, directly inhibits pathogens.We found that ergosterol and the Prb1 protein strongly bind to each other, but ergosterol and the OsZF8-Prb1 protein complex only weakly bind.In summary, OsZF8 inhibits the binding of the Prb1 protein to ergosterol by binding the Prb1 protein and negatively regulates rice resistance to R. solani.Based on the above results, it is speculated that although some fungi can synthesize ergosterol by themselves, the binding efficiency of PRB-1 to ergosterol is higher than its autosynthesis rate, and thus has a suppression on fungi.and the overexpressing mutant OsZF8 OX were planted in the greenhouse of Shenyang Agricultural University.The R. solani strain AG1-IA was cultured on solid PDA (potato dextrose agar) medium at 28 • C in an incubator.The inoculation method referred to the study by Cao et al. [42].The rice cDNA library plasmid, pGBKT7 vector, pGADT7 vector, pSPYNE vector, pSPYCE vector, pINDEX2 vector, and pCBDEST vector were all sourced from the College of Plant Protection, Shenyang Agricultural University.

Materials and Methods
OsZF8-ko12 plants and OsZF8 OX plants were grown for 60 days; WT plants were grown for 90 days.The roots, stems, leaves, leaf sheaths, and ears of the WT plants were collected as samples for the analysis of OsZF8 expression in different rice organs (OsZF8F/OsZF8R).The Y-36 strain was inoculated onto isolated leaves of inverted two-leaf plants (60 days), and samples were collected at 0 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, and 96 h for analysis of the expression of OsZF8 in response to infection by Y-36.OsZF8-ko12 and OsZF8 OX plants were used as mutant plants for verification.The expressions of NPR1, EIN2, EIL1, D61, D2, GID1, and SLR1 in WT, OsZF8-ko12, and OsZF8 OX plants were analyzed 48 h after inoculation with the Y-36 strain.The primers used were as follows: qNPR1F/qNPR1R, qEIN2F/qEIN2R, qEIL1F/qEIL1R, qD61F/qD61R, qD2F/qD2R, qGID1F/qGID1R, and qSLR1F/qSLR1R.The expressions of PBZ1 and PR1b in WT, OSZF8-ko12, and OsZF8 OX plants were analyzed 72 h after inoculation with the Y-36 strain.The primers were PBZ1F/PBZ1R and PR1BF/PR1BR.In all experiments involving OsZF8 OX plants, 20 µM dexamethasone (DEX) was used to spray 24 h before the experiment.All primers are shown in Table S1.

Construction of OsZF8 Mutant Plants
The OsZF8 knockout mutant-clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-was used to construct the OsZF8 knockout construct; the target sequence was designed, and BGE Gene Technology Co., Ltd.(Changzhou, Jiangsu, China) constructed the knockout mutant.With the use of hygromycin screening and sequencing, the transgene was verified, and a positive T0 generation knockout mutant was obtained.The primers used were SpeI-ZF8F/SpeI-ZF8R.After the single T0 knockout mutant plant was propagated, the T1 knockout plant was planted, and sequencing and qPCR verification were performed again.The primers used were ZF8qF/ZF8qR.
OsZF8 overexpression mutant: Rice cDNA was used as the template for the amplification of OsZF8, and after the amplified fragment was recovered, the enzyme digestion product was ligated to the pINDEX2-FLAG vector via SpeI and XhoI sites.The recombinant protein was subsequently transformed into DH5α cells, after which the plasmid was extracted for sequencing.The primers used were ZF8-qF/M13R.The correct overexpression vector was sequenced and transformed into the EHA105 strain.Positive clones were selected, cultured, and subsequently sent to Baige Gene Technology Co., Ltd.(Changzhou, Jiangsu, China) for overexpressing plant construction.Positive T0 mutant plants were obtained via hygromycin screening.The primers used were ZF8-qF/M13R.Transformed plants with the correct target band size were selected for breeding, and the expression level of ZF8 in T1 generation plants was detected by qPCR.The primers used were ZF8qF/ZF8qR.

RNA Extraction and Quantitative Real-Time (qRT)-PCR Analysis
Briefly, 0.1 g of inoculated sample was fully ground with liquid nitrogen for RNA extraction and cDNA synthesis according to the instructions for the Ultrapure RNA Kit Extraction Kit (CWBIO, CW0581, Taizhou, Jiangsu, China) and HiScript III RT SuperMix for qPCR Kit (Vazyme, R323-01, Nanjing, Jiangsu, China).ChamQ Universal SYBR qPCR Master Mix (Vazyme, Q711) was used to configure the reaction system, and real-time fluorescent quantitative PCR was performed using a CFX Duet fluorescent quantitative PCR instrument (Bio-Rad, Hercules, CA, USA).Two technical and three biological replicates were used for each analysis.The fluorescence signal was collected after the annealing/extension step.Ct values were determined after the reaction, and the data were analyzed using the 2 −∆∆Ct method [43].

Yeast Two-Hybrid Assay
A cDNA library of rice was screened with pGBKT7-OsZF8 as bait and via the mating method.The CDS of the interacting protein PRB1 was obtained, rice cDNA was used as the template, the target protein CDS was cloned, and the sequence was inserted into pGADT7 via enzyme digestion.After enzyme digestion and sequencing verification, the AD and BD plasmids were transferred to the Y2H strain, which was subsequently cultured on SD/-Trp/-Leu plates.Positive colonies were selected and diluted according to the gradient, and SD/-Trp/-Leu/-His/-Ade/X-alpha-gal/AbA was used for detection.Primers (ADF/ADR, AD-PRB1F/AD-PRB1R) are shown in Table S1.

Bimolecular Fluorescence Complementation Assay
The pSPYNE-OsZF8 plasmid and pSPYCE-AD plasmid were constructed via homologous recombination and subsequently transferred to Agrobacterium EHA105.PCR was used to amplify the NbH2B fragment harboring the attB1 and attB2 sequences at both ends (the stop codon was removed).The NbH2B gene fragment was subsequently transferred into the pH7RWG2.0vector.Strains carrying pSPYNE-OsZF8, pSPYCE-AD, and pH7RWG2.0-H2Bwere infiltrated and inoculated onto tobacco leaves for 30 days, after which fluorescence was observed under a 488-550 mm laser confocal microscope after 48 h.

Instantaneous Expression of Tobacco
The protein-coding sequences of OsZF8 and Prb1 were reconstructed to the pCBDESTtransient sequence using the Gateway method; GFP and BAX were used as negative and positive controls, respectively.The obtained plasmid was subjected to thermal shock transformation using the competent cell line GV3101, which was subsequently coated on a plate containing LB + Rif + Kana for positive cloning screening.The positive clone was added to 2 mL of liquid LB (containing Rif + Kana) and shaken at 28 • C and 220 r•min −1 for 16 h, and the culture was diluted to a final concentration of 0.5 optical density (OD).Tobacco cultured for 30 days was used in the test.The penetration solution was gently injected into tobacco cells from the back of the leaves using a 1 mL sterile syringe.The penetration diameter of each injection site was approximately 1.5 cm.The surface of the leaves was wiped dry.After 3 days of culture, the degree of cell death was observed and photographed [44,45].

Molecular Docking
ASphafold 2 software was used to predict the crystal structure of the Prb1 and OsZF8 proteins.PyMol 2.5.3 software removed water molecules, hydrogen atoms, and nontarget structural proteins from the PRB1 and OsZF8 proteins.ZDOCK 3.0.2software was employed to simulate the binding mode of PRB1 and OsZF8.After docking, AMBER 18 software was used to minimize the energy under the ff14SB force field, and Prodigy software (v1.12.0) was used to evaluate the binding energy.PyMol 2.5.4 software was used for visual analysis based on the optimal binding energy binding mode [46].
Prb1 and OsZF8-Prb1 protein complexes were derived from the above prediction and bonding results.The 3D structure of ergosterol (CAS: 57-87-4) was downloaded from the PUBCHEM database and minimized under the MMFF94 force field.AutoDock Vina 1.1.2software was used to dock proteins with small molecules, and PyMol 2.5 software was used to analyze the docking results visually [47,48].

Statistical Analyses
The structure and functional domains of the OsZF8 protein were analyzed via NCBI-SMART online software (https://blast.ncbi.nlm.nih.gov/smartblast/?LINK_LOC=Blas tHomeLink, accessed on 24 May 2024), and a phylogenetic tree of the OsZF8 protein was constructed via MEGA5.0software.Statistical analyses were performed using Microsoft Office Excel 2016, and significance analysis was performed using SPSS 22.0.Also, Student's t-test was used to compare the differences between the two groups.Differences between the groups were considered significant with at least p < 0.05.

Conclusions
In summary, OsZF8 was significantly up-regulated after R. solani AG1-IA inoculation, suggesting that OsZF8 plays an important regulatory role in the interaction between rice and Rhizoctonia solani.In this study, OsZF8 expression pattern, mutant phenotype identification, bioinformatics, and expression of related defense genes were analyzed.Transient expression, yeast two-hybrid, and molecular docking were used to analyze the regulatory function of OsZF8 at post-transcriptional protein level.OsZF8 inhibits the binding ability of Prb1 protein to ergosterol by binding to Prb1 protein, and negatively regulates rice resistance to sheath blight.This results provided new insights for understanding the mechanism of resistance to rice sheath blight.

Figure 1 .
Figure 1.Expression analysis of OsZF8.(A): Expression analysis of OsZF8 in response to inf by Y-36 strain.The expression level of OsZF8 reached its highest 60 h after inoculation.(B): Ex sion analysis of OsZF8 in different parts of rice.The expression of OsZF8 was highest in roo lowest in panicles.Error bars represent means ± SE of three repeated experiments (n = 3).Diff letters indicate significant differences (p < 0.05).

Figure 2 .
Figure 2. Domain and phylogenetic analysis of the OsZF8.(A): Phylogenetic analysis of OsZF numbers indicate the accession numbers of gene sequences of the species in GenBank.(B): P domain analysis of OsZF8.

Figure 1 .
Figure 1.Expression analysis of OsZF8.(A): Expression analysis of OsZF8 in response to infection by Y-36 strain.The expression level of OsZF8 reached its highest 60 h after inoculation.(B): Expression analysis of OsZF8 in different parts of rice.The expression of OsZF8 was highest in roots and lowest in panicles.Error bars represent means ± SE of three repeated experiments (n = 3).Different letters indicate significant differences (p < 0.05).

Figure 1 .
Figure 1.Expression analysis of OsZF8.(A): Expression analysis of OsZF8 in response to infection by Y-36 strain.The expression level of OsZF8 reached its highest 60 h after inoculation.(B): Expression analysis of OsZF8 in different parts of rice.The expression of OsZF8 was highest in roots and lowest in panicles.Error bars represent means ± SE of three repeated experiments (n = 3).Different letters indicate significant differences (p < 0.05).

Figure 2 .
Figure 2. Domain and phylogenetic analysis of the OsZF8.(A): Phylogenetic analysis of OsZF8.The numbers indicate the accession numbers of gene sequences of the species in GenBank.(B): Protein domain analysis of OsZF8.

Figure 2 .
Figure 2. Domain and phylogenetic analysis of the OsZF8.(A): Phylogenetic analysis of OsZF8.The numbers indicate the accession numbers of gene sequences of the species in GenBank.(B): Protein domain analysis of OsZF8.

Figure 3 .
Figure 3. OsZF8 mutants are more resistant to sheath blight.(A): Expression analysis of OsZF8 in OsZF8 OX1 and OsZF8 OX2 plants.(B): Expression analysis of OsZF8 in OsZF8-ko12 and OSZF8-ko29 plants.(C): Insertion site of OsZF8 knockout mutant.Blue and white boxes indicate exons and untranslated regions, respectively.The red triangle represents the editing site.(D): Construction of OsZF8 overexpression vector.* represents concatenation of different numbers of tags.(E): Relative lesion area on leaves shown in (G) leaves inoculated with R. solani AG1-IA.(F): Relative biomass shown in (G) leaves inoculated with R. solani AG1-IA.(G): The pathogenic symptoms of OsZF8-ko12 and OsZF8 OX plants.ns, no significant difference.p-values were analyzed using student's t-test (p *** < 0.001).

Figure 3 .
Figure 3. OsZF8 mutants are more resistant to sheath blight.(A): Expression analysis of OsZF8 in OsZF8 OX1 and OsZF8 OX2 plants.(B): Expression analysis of OsZF8 in OsZF8-ko12 and OSZF8-ko29 plants.(C): Insertion site of OsZF8 knockout mutant.Blue and white boxes indicate exons and untranslated regions, respectively.The red triangle represents the editing site.(D): Construction of OsZF8 overexpression vector.* represents concatenation of different numbers of tags.(E): Relative lesion area on leaves shown in (G) leaves inoculated with R. solani AG1-IA.(F): Relative biomass shown in (G) leaves inoculated with R. solani AG1-IA.(G): The pathogenic symptoms of OsZF8-ko12 and OsZF8 OX plants.ns, no significant difference.p-values were analyzed using student's t-test (p *** < 0.001).

Figure 5 .
Figure 5. OsZF8 interacts with Prb1.(A): Yeast two-hybrid assay for OsZF8 interactions with Prb1.(B): Bimolecular fluorescence complementation assay for OsZF8 and Prb1 interaction.pSPYNE-OsZF8, pSPYCE-Prb1 were co-expressed in tobacco leaves.Bar = 50 µm.(C): OsZF8 suppresses PRB1-triggered cell death in tobacco.The Agrobacterium strain carrying OsZF8 + PRB1, and gfp gene construct were co-infiltrated with Prb1 gene construct into the right leaves, respectively.The Agrobacterium strain carrying Prb1 and OsZF8 gene construct was infiltrated into the left leaves.Cell death symptoms on the infiltrated leaves were photographed.

Figure 5 .
Figure 5. OsZF8 interacts with Prb1.(A): Yeast two-hybrid assay for OsZF8 interactions with Prb1.(B): Bimolecular fluorescence complementation assay for OsZF8 and Prb1 interaction.pSPYNE-OsZF8, pSPYCE-Prb1 were co-expressed in tobacco leaves.Bar = 50 µm.(C): OsZF8 suppresses PRB1-triggered cell death in tobacco.The Agrobacterium strain carrying OsZF8 + PRB1, and gfp gene construct were co-infiltrated with Prb1 gene construct into the right leaves, respectively.The Agrobacterium strain carrying Prb1 and OsZF8 gene construct was infiltrated into the left leaves.Cell death symptoms on the infiltrated leaves were photographed.

Figure 6 .
Figure 6.Binding mode of OsZF8 and Prb1 protein.The wheat color in the figure represents the OsZF8 protein model, the blue color represents the Prb1 protein model, and the dashed yellow line represents hydrogen bonding.The following figure uses the same color coding.

Figure 7 .
Figure 7. Binding mode of Prb1 protein and OsZF8-Prb1 protein complex with ergosterol (A): The binding mode of Prb1 protein and ergosterol.(B): Binding mode of OsZF8-Prb1 protein complex and ergosterol.The wheat color in the figure represents the OsZF8 protein model, the blue color represents the Prb1 protein model, and the dashed yellow line represents hydrogen bonding.The following figure uses the same color coding.Ergosterol has a hydrophobic effect on ASN-144, PRO-159, TRP-115, GLN-146, and TRP-104 of the OsZF8-Prb1 protein.This leads to the combination capacity of ergosterol and OsZF8-PRB1 protein being weak.

Figure 6 .
Figure 6.Binding mode of OsZF8 and Prb1 protein.The wheat color in the figure represents the OsZF8 protein model, the blue color represents the Prb1 protein model, and the dashed yellow line represents hydrogen bonding.The following figure uses the same color coding.

Figure 6 .
Figure 6.Binding mode of OsZF8 and Prb1 protein.The wheat color in the figure represents the OsZF8 protein model, the blue color represents the Prb1 protein model, and the dashed yellow line represents hydrogen bonding.The following figure uses the same color coding.

Figure 7 .
Figure 7. Binding mode of Prb1 protein and OsZF8-Prb1 protein complex with ergosterol (A): The binding mode of Prb1 protein and ergosterol.(B): Binding mode of OsZF8-Prb1 protein complex and ergosterol.The wheat color in the figure represents the OsZF8 protein model, the blue color represents the Prb1 protein model, and the dashed yellow line represents hydrogen bonding.The following figure uses the same color coding.Ergosterol has a hydrophobic effect on ASN-144, PRO-159, TRP-115, GLN-146, and TRP-104 of the OsZF8-Prb1 protein.This leads to the combination capacity of ergosterol and OsZF8-PRB1 protein being weak.

Figure 7 .
Figure 7. Binding mode of Prb1 protein and OsZF8-Prb1 protein complex with ergosterol (A): The binding mode of Prb1 protein and ergosterol.(B): Binding mode of OsZF8-Prb1 protein complex and ergosterol.The wheat color in the figure represents the OsZF8 protein model, the blue color represents the Prb1 protein model, and the dashed yellow line represents hydrogen bonding.The following figure uses the same color coding.Ergosterol has a hydrophobic effect on ASN-144, PRO-159, TRP-115, GLN-146, and TRP-104 of the OsZF8-Prb1 protein.This leads to the combination capacity of ergosterol and OsZF8-PRB1 protein being weak.

4 . 1 .
Plant Growth and R. solani Inoculation R. solani AG1-IA (Y-36), Y2H strains, Y187 strains, DH5α, EHA105 strains, and GV3101 strains were preserved at the College of Plant Protection, Shenyang Agricultural University (Shenyang, Liaoning, China).All of the rice plants treated with R. solani were cultured in the Shenyang Agriculture University greenhouse at 23-30 • C, 80% relative humidity (RH), and 12-h light/12-h dark photoperiod.Tobacco was cultured at 25 • C under 16 h of light and at 23 • C under 8 h of darkness.The OsZF8 knockout mutant OsZF8-ko12 Author Contributions: Y.W.(Yan Wang), S.W. and H.W. designed the project and wrote the manuscript.Y.W. (Yan Wang), L.Z., Y.W.(Yiming Wang), H.W. and L.W. performed the experiments and analyzed the data.All authors have read and agreed to the published version of the manuscript.