Comprehensive Analysis of Autophagy-Related Genes in Rice Immunity against Magnaporthe oryzae

Rice blast disease, caused by the fungus Magnaporthe oryzae, is a significant threat to rice production. Resistant cultivars can effectively resist the invasion of M. oryzae. Thus, the identification of disease-resistant genes is of utmost importance for improving rice production. Autophagy, a cellular process that recycles damaged components, plays a vital role in plant growth, development, senescence, stress response, and immunity. To understand the involvement of autophagy-related genes (ATGs) in rice immune response against M. oryzae, we conducted a comprehensive analysis of 37 OsATGs, including bioinformatic analysis, transcriptome analysis, disease resistance analysis, and protein interaction analysis. Bioinformatic analysis revealed that the promoter regions of 33 OsATGs contained cis-acting elements responsive to salicylic acid (SA) or jasmonic acid (JA), two key hormones involved in plant defense responses. Transcriptome data showed that 21 OsATGs were upregulated during M. oryzae infection. Loss-of-function experiments demonstrated that OsATG6c, OsATG8a, OsATG9b, and OsATG13a contribute to rice blast resistance. Additionally, through protein interaction analysis, we identified five proteins that may interact with OsATG13a and potentially contribute to plant immunity. Our study highlights the important role of autophagy in rice immunity and suggests that OsATGs may enhance resistance to rice blast fungus through the involvement of SA, JA, or immune-related proteins. These findings provide valuable insights for future efforts in improving rice production through the identification and utilization of autophagy-related genes.


Introduction
Autophagy is a cellular recycling mechanism that involves the degradation and recycling of damaged or unnecessary cellular components, such as proteins, organelles, and pathogens, to maintain cellular homeostasis and promote cell survival [1,2].Based on the different degradation targets and degradation mechanisms, autophagy can be divided into three types: macroautophagy, microautophagy, and selective autophagy [1].The most commonly referenced type is macroautophagy, wherein the substrates to be degraded are enveloped by structures called autophagosomes and transported to lysosomes or vacuoles for degradation [3].The process of autophagy can be further subdivided into several stages, namely induction, nucleation, elongation, closure, transport, and degradation [4].
Plants 2024, 13, 927 2 of 20 Autophagy-related genes (ATGs) are a group of genes involved in the autophagy process.They are responsible for functions such as forming the autophagosome membrane, recruiting cargo to the autophagosome, and facilitating the fusion of autophagosomes with lysosomes for degradation [5].These ATGs are highly conserved across different species, including plants, animals, and fungi.The first ATG was identified in Saccharomyces cerevisiae, and since then, over 40 ATGs have been identified in eukaryotic organisms [6,7].In various plant model organisms, such as Arabidopsis thaliana, Zea mays, Oryza sativa, Rosa roxburghii, and Camellia sinensis, ATGs have been extensively studied and found to be involved in growth, development, senescence, stress response, and immunity [8][9][10][11].
Autophagy plays a crucial role in the growth and development of plants.ATGs have been identified as key players in the aging process in various plant species, including Arabidopsis thaliana, Zea mays, Hordeum vulgare, Glycine max, Petunia hybrida, and Brassica rapa [8,[12][13][14][15][16].In Arabidopsis, ATGs are known to regulate the production of reactive oxygen species (ROS) and indole-3-acetic acid (IAA), thus influencing glucose-mediated root meristem growth [17].Additionally, the knockout of OsATG7 in rice has been shown to have a significant impact on seed size and starch content, highlighting the essential role of autophagy in seed development [18].
Autophagy works as a fundamental defense response and stress regulatory mechanism that promotes cell repair and regeneration.When plants are subjected to biotic and abiotic stresses, autophagy is significantly activated to facilitate organismal repair [19].Defects in autophagy make plants more susceptible to abiotic stress [20].For instance, the silencing of Arabidopsis AtATG18 leads to increased drought sensitivity [21].On the other hand, the overexpression of MdATG18 in Malus domestica enhances apple tolerance to drought stress [22].Under cold stress, the expression of rice OsATG6a/c is downregulated [23], while barley HvATG6 is upregulated [24].Moreover, the functional deficiency of wheat TaATG7 and rice OsATG10b reduces plant tolerance to salt stress [25,26], whereas the overexpression of MdATG10 in apples increases their tolerance to salt stress [27].
Autophagy plays a crucial role in plant immunity and defense against pathogen invasion [28].For example, AtATG18a interacts with AtWRKY33 to aid plants in defending against necrotrophic pathogens [29].In Solanum lycopersicum, the silencing of SlATG5, SlATG7, and SlATG8h promotes infection by TLCYnV (tomato leaf curl Yunnan virus) [30].Similarly, the loss of function of OsATG5 or OsATG7 in rice leads to increased susceptibility to RSV (rice stripe virus), highlighting the role of autophagy in suppressing RSV infection [31].
Rice blast, caused by the fungus Magnaporthe oryzae, is a severe disease that greatly impacts rice production and global yield.However, the understanding of how rice autophagy, an essential plant immunity regulatory mechanism, contributes to rice resistance against blast disease remains limited.In 2011, Xia et al. discovered 33 ATGs in rice, marking an early milestone in autophagy research [8].Since then, with advancements in autophagy research and the refinement of rice gene annotation, more OsATGs (ATGs in O. sativa) have been identified.Building upon the foundation laid by Xia et al., we further refined the list by excluding five OsATGs lacking the ATG domain or gene ID.Moreover, we included nine OsATGs that exhibited ATG-related structural domains.The primary objective of this study is to investigate the role of 37 OsATGs in rice immunity against blast disease.

Physicochemical Properties and Subcellular Localization of OsATGs
As shown in Table 1, different OsATG proteins exhibit varying physicochemical properties.The amino acid count ranges from 93 to 1919, while the molecular weights range from 10,461.01 to 212,230.11Da.The theoretical pI (isoelectric point) ranges from 4.52 (OsATG3a) to 9.71 (OsATG14b), with an average of 6.81.Out of the 37 OsATG proteins, 12 are considered alkaline (pI > 7.5), while 25 are considered acidic (pI < 7.5).Furthermore, thirty OsATG proteins are stable (with an instability index exceeding 40, which is the threshold for stable proteins), while the remaining seven are unstable.The lipid coefficient (shown as the aliphatic index) ranges from 65 to 102.36.Hydrophobicity analysis showed that thirty-six OsATG proteins are hydrophilic (GRAVY < 0), while only one is hydrophobic (GRAVY > 0).The prediction of protein subcellular localization showed that these OsATG proteins primarily reside in the nucleus, inner membrane, and organelle membranes.Additionally, we performed tertiary structure analyses of these OsATG proteins and found that different homologous proteins of the same OsATG have similar protein conformations (Figure S1).

Phylogenetic Relationship and Chromosomal Localization of OsATGs
To investigate the phylogenetic relationship of OsATG proteins, we performed cluster analysis using the neighbor-joining method in MEGA11.0 software.The results revealed that OsATG proteins can be divided into five categories (Figure 1a).Eight OsATG proteins, including OsATG2, OsATG5, OsATG7, OsATG11, OsATG12, OsATG14a, OsATG14b, and OsATG16, belong to distinct evolutionary branches, indicating their independent evolution.The remaining 29 OsATG proteins can be further divided into groups I, II, III, and IV, with variations in the number of proteins in each group.Group I consists solely of three OsATG13 proteins; group II consists solely of seven OsATG18 proteins; group III includes two OsATG3 proteins, two OsATG4 proteins, three OsATG6 proteins, and two OsATG10 proteins; the remaining ten OsATG proteins are clustered in group IV.These findings provide insights into the evolutionary relationships and diversification of OsATG proteins.
Furthermore, we analyzed the chromosomal localization of OsATGs.The results showed that the 37 OsATGs are distributed unevenly across 11 chromosomes, excluding Chr 9 (Figure 1b).Chr 3 contains the highest number of genes (seven), followed by Chr 1 (six), while Chr 8 and Chr 12 have the fewest genes (one each).Interestingly, we found that approximately one-third of the genes are located near the telomeric regions of the chromosomes, while the majority of the remaining genes are situated in the distal regions away from the telomeres.These findings provide valuable insights into the spatial distribution patterns of OsATGs on the rice chromosomes.

Gene Structure and Function Domain of OsATGs
Analysis of the gene structure of OsATGs revealed variations in the number of exons among the members (Figure 2a).Notably, OsATG6c has the fewest with one exon, while OsATG7 has the most with twenty exons.The remaining genes have varying numbers of exons ranging from two to thirteen.This uneven distribution of exons suggests potential intron insertion or deletion events during the evolution of OsATGs.Further examination of the functional domains of OsATGs unveiled that all members harbor typical autophagyrelated domains, as illustrated in Figure 2b.Specifically, three OsATG1s feature a kinase domain, while one OsATG2, two OsATG3s, and two OsATG10s contain an ATG domain at the C-terminal region.Additionally, two OsATG4s possess a Peptidase-C54 domain, and all seven OsATG18s include a WD40 domain.The remaining OsATGs each contain a specific autophagy-related domain.These findings suggest that OsATGs are involved in a diverse array of biological processes.

Gene Structure and Function Domain of OsATGs
Analysis of the gene structure of OsATGs revealed variations in the number of exons among the members (Figure 2a).Notably, OsATG6c has the fewest with one exon, while OsATG7 has the most with twenty exons.The remaining genes have varying numbers of exons ranging from two to thirteen.This uneven distribution of exons suggests potential intron insertion or deletion events during the evolution of OsATGs.Further examination of the functional domains of OsATGs unveiled that all members harbor typical autophagy-related domains, as illustrated in Figure 2b.Specifically, three OsATG1s feature a kinase domain, while one OsATG2, two OsATG3s, and two OsATG10s contain an ATG domain at the C-terminal region.Additionally, two OsATG4s possess a Peptidase-C54 domain, and all seven OsATG18s include a WD40 domain.The remaining OsATGs each contain a specific autophagy-related domain.These findings suggest that OsATGs are involved in a diverse array of biological processes.

Protein Motif of OsATGs
Motif prediction revealed that, among 37 OsATG proteins, 26 contain motifs, with a total of 20 motifs identified (Figure 3).These motifs vary in terms of number, type, and length.The shortest motif (motif 12) is only 21 amino acids long and is present in five OsATG8 proteins.On the other hand, the longest motifs (motifs 1-5, 7, 8, 14, 19, and 20) span 50 amino acids and are found in ten OsATG proteins.All 26 OsATG proteins contain these longest motifs, except for OsATG13a, OsATG13b, and OsATG16.Interestingly, it was observed that three OsATG1 proteins and three OsATG6 proteins contain motif 5.

Protein Motif of OsATGs
Motif prediction revealed that, among 37 OsATG proteins, 26 contain motifs, with a total of 20 motifs identified (Figure 3).These motifs vary in terms of number, type, and length.The shortest motif (motif 12) is only 21 amino acids long and is present in five OsATG8 proteins.On the other hand, the longest motifs (motifs 1-5, 7, 8, 14, 19, and 20) span 50 amino acids and are found in ten OsATG proteins.All 26 OsATG proteins contain these longest motifs, except for OsATG13a, OsATG13b, and OsATG16.Interestingly, it was observed that three OsATG1 proteins and three OsATG6 proteins contain motif 5. Additionally, motif 1 is present in five OsATG8 proteins, as well as OsATG9a and OsATG11.motif 11 is found exclusively in OsATG13a and OsATG13b.Furthermore, all five OsATG18 proteins contain motif 3. Notably, OsATG6a, OsATG6b and OsATG6c have the highest number of motifs, suggesting their involvement in multiple pathways.These findings indicate that OsATGs can be regulated though various motifs, allowing them to serve diverse functions within the organism.

Genetic cis-Regulatory Elements of OsATGs
The analysis of cis-regulatory elements (CREs) was performed on the 2 kb upstream promoter sequence of OsATGs.The results revealed that there are 24 types of CREs in the promoter region (Figure 4a).The study primarily focused on the involvement of these CREs in hormone regulation and stress response.The CREs of hormone regulation include response to salicylic acid (SA), methyl jasmonate (MeJA), ethylene (ETH), abscisic acid (ABA), gibberellin (GA), and auxin (AUX).And the CREs of stress response include responses to low temperature, drought, wounding, and defense.Analyzing the hormone regulatory CREs, we found that the promoter region of 13 OsATG genes contain CREs of SA response, and 30 OsATGs contain MeJA, 16 OsATGs contain ETH, 34 OsATGs contain ABA, 20 OsATGs contain GA, 17 OsATGs contain AUX (Figure 4b).This significant enrichment of hormone CREs suggests that OsATGs play crucial roles in various biological processes mediated by different plant hormones.responses to low temperature, drought, wounding, and defense.Analyzing the hormone regulatory CREs, we found that the promoter region of 13 OsATG genes contain CREs of SA response, and 30 OsATGs contain MeJA, 16 OsATGs contain ETH, 34 OsATGs contain ABA, 20 OsATGs contain GA, 17 OsATGs contain AUX (Figure 4b).This significant enrichment of hormone CREs suggests that OsATGs play crucial roles in various biological processes mediated by different plant hormones.

Resistance to Rice Blast Fungus of OsATGs
Using the available transcriptome data during infection by M. oryzae in rice, a hierarchical clustering heatmap was generated to analyze the expression patterns of OsATGs.Out of the 37 OsATG genes analyzed, with the exception of OsATG8e and OsATG18g, all were expressed and showed differential expression patterns (Figure 5a and Table S2).The results also showed that 17 OsATGs exhibited increased expression at 24 h post-infection, while 10 OsATGs showed increased expression at 48 h.Notably, six OsATGs (OsATG1b, OsATG6a, OsATG6c, OsATG10a, OsATG12, and OsATG13b) displayed sustained increased expression throughout the 0-48 h period.These results strongly suggest that autophagy plays a significant role in conferring resistance to rice blast fungus.To further investigate the involvement of OsATGs in rice immunity against blast fungus, we conducted a disease resistance analysis on six loss-of-function mutants of OsATGs (OsATG6b, OsATG6c, OsATG8a, OsATG9a, OsATG9b, and OsATG13a).The results of inoculating rice with M. oryzae showed that the relative lesion area of the osatg8a and osatg13a mutants significantly increased compared to the wild type, while there was no significant change in the other mutants (Figure 5b).Interestingly, when wounded rice was inoculated, the osatg6c and osatg9b mutants also showed enhanced susceptibility to M. oryzae (Figure 5c).These results indicate that OsATG6c, OsATG8a, OsATG9b, and OsATG13a play a positive role in regulating resistance to rice blast.The qRT-PCR results showed that OsATG6b and OsATG9a were significantly upregulated at various time points during M. oryzae infection (Figure 5d), suggesting that they are also involved in responding to M. oryzae infection.Taken together, these findings provide compelling evidence that rice OsATGs play a crucial role in immunity against blast fungus.

Response to Hormone Treatments of OsATGs
In order to investigate whether OsATGs are involved in the SA and JA signaling pathways, we analyzed the expression levels of OsATG6b, OsATG6c, OsATG8a, OsATG9a, OsATG9b, and OsATG13a genes in the wild-type ZH11 after induction with SA and MeJA at 0, 6, 12, and 36 h.The results showed that five OsATG genes (OsATG6b, OsATG6c, Os-ATG8a, OsATG9b, and OsATG13a) were responsive to the SA signaling pathway (Figure 6a), while five OsATG genes (OsATG6c, OsATG8a, OsATG9a, OsATG9b, and OsATG13a) were responsive to the SA signaling pathway (Figure 6b).Under SA induction, OsATG6b and OsATG8a were rapidly upregulated at 6 h, indicating their potential involvement in the rapid response to the SA pathway.OsATG9b and OsATG13a were upregulated from 12 to 36 h, while the expression level of OsATG6c decreased at 12 h.Similarly, under JA induction, the expression levels of four genes (OsATG6c, OsATG8a, OsATG9b, and OsATG13a) significantly increased at 6 h, demonstrating a rapid response to the JA signal, while the expression level of OsATG9a decreased at 36 h.Based on the above data, we hypothesize that OsATGs may participate in rice growth, development, and stress response mediated by SA or JA signaling pathways.

Subcellular Localization and Role of Immune Reaction of OsATG13a
Based on our analysis of pathogenicity and gene expression patterns induced by the plant hormones SA and JA, we have discovered that OsATG13a plays an important role in rice resistance against the blast fungus.To further investigate its function, we conducted a subcellular localization assay on OsATG13a.The results showed that OsATG13a was localized in the cytoplasm, with a strong punctate localization (Figure 7a).This punctate localization is consistent with the positioning of ATG16 in the phagosome [32].Furthermore, our investigation revealed that GFP-OsATG13a co-localized with RFP-ATG16, confirming that the strong punctate localization of OsATG13a corresponds to the phagosome (Figure 7b).
Additionally, we examined the effects of chitin treatment on callose accumulation and the burst of ROS in the osatg13a mutant.Our results demonstrate that the osatg13a mutant exhibits significantly lower levels of both callose accumulation and ROS burst compared to the wild type under chitin induction (Figure 7c,d).These findings suggest that the loss function of OsATG13a leads to a reduced PTI response in rice against the blast fungus.

Possible Interaction Proteins of OsATG13a
We conducted a yeast two-hybrid screen to identify potential interacting proteins of OsATG13a.From this screen, we obtained a total of 104 transformants.After sequencing and verification, we identified 50 proteins that may interact with OsATG13a (Figure 8a and Supplementary Table S1).To further understand the roles of these proteins, we conducted a Gene Ontology (GO) analysis and found that they are involved in catalytic activity, cellular metabolism, and structural formation (Figure 8b).Notably, among these proteins, we discovered that five, namely OsRLK170576, OsKEG1, OsANR1, OsPP2A-4, OsVPE1, and OsMDH1, are potentially associated with plant immune responses (Table 2).This suggests that OsATG13a may play a role in the immune processes of rice through its interaction with these proteins.
firming that the strong punctate localization of OsATG13a corresponds to the phagosome (Figure 7b).
Additionally, we examined the effects of chitin treatment on callose accumulation and the burst of ROS in the osatg13a mutant.Our results demonstrate that the osatg13a mutant exhibits significantly lower levels of both callose accumulation and ROS burst compared to the wild type under chitin induction (Figure 7c,d).These findings suggest that the loss function of OsATG13a leads to a reduced PTI response in rice against the blast fungus.ducted a Gene Ontology (GO) analysis and found that they are involved in catalytic activity, cellular metabolism, and structural formation (Figure 8b).Notably, among these proteins, we discovered that five, namely OsRLK170576, OsKEG1, OsANR1, OsPP2A-4, OsVPE1, and OsMDH1, are potentially associated with plant immune responses (Table 2).This suggests that OsATG13a may play a role in the immune processes of rice through its interaction with these proteins.

Identification of OsATGs in Rice
A BLAST search was conducted in NCBI to identify ATG homologs in rice, with Arabidopsis AtATGs as the reference.It is to be noted that the search was specifically confined to the Oryza sativa Japonica Group (taxid: 39,947).A second round of searching was performed using the identified rice ATG homologs to ensure comprehensive coverage, while maintaining focus on the Oryza sativa Japonica Group (taxid: 39,947).The ATG gene IDs in three databases and the functional domains in NCBI were also searched.Detailed information on OsATGs is provided as Supplementary Table S1.

Bioinformatics Characterization Analysis
The mRNA and chromosome positions of OsATGs were obtained from the NCBI website.The Protparam website was used to calculate the physicochemical properties of OsATGs [39].The Busca website was employed to predict the subcellular localization of OsATGs [40].The functional domains of OsATGs were obtained from the SMART website [41].The MEME website was used to identify conserved motifs of OsATGs [42].The PlantCARE website was utilized to identify cis-acting elements in the upstream 2 kb promoter regions of OsATGs [43].Data visualization was performed using the TBtools v1.098 software.The MEGA v11.0 software was used to construct a phylogenetic tree using the neighbor-joining method [44], and the tree was subsequently modified and refined using the Evolview website [45].The SWISS-MODEL website was employed to predict the 3D structure of the proteins encoded by OsATGs [46].The online URLs for all websites can be found in Supplementary Table S5.

Analysis of Gene Expression Patterns
Rice leaves were collected at 0 h, 24 h, and 48 h after infection with the rice blast fungus.The collected leaves were sent to Igenebook (Wuhan igenebook Biotechnology Co., Ltd., Wuhan, China) for RNA-seq analysis.The company performed expression analysis of various genes in rice.Subsequently, the expression levels of OsATG genes in rice were obtained and visualized using TBtools software.

Plants and Materials
All experiments were performed using Oryza sativa L. ssp.Japonica cv.Zhonghua 11.Mutants of rice were generously provided by Prof. Qingjun Xie (South China Agricultural University, Guangzhou, China).The specific mutation sites of each mutant can be found in Supplementary Figure S2.Unless otherwise specified, the rice plants were cultivated under the following conditions: a temperature of 26 • C, relative humidity of 50%, light intensity of 200 µmol m −2 s −1 , and a 12 h photoperiod.Plasmids pHF223 and pHF225 were utilized for the construction of GFP or RFP fusion proteins, and the primer sequences can be found in Supplementary Table S1.For pathogen treatment, a concentration of 1-50 × 10 5 mL −1 of rice blast fungus conidial suspension was employed.Regarding hormone treatment, SA and MeJA were added at concentrations of 1 mmol and 0.1 mmol, respectively, and the solvents used were sterile water containing 0.01% (v/v) Tween 20.

Disease Resistance Analysis
For spray inoculation, a 5 mL M. oryzae conidial suspension, containing 1 × 10 4 mL −1 , was evenly sprayed onto the 2-week-old rice seedlings using a sprayer pump bottle.For wound inoculation, the leaves of 8-week-old rice plants were gently punctured and then injected with 0.007 mL conidial suspension.Post inoculation, the rice plants were sealed for 24 h [47], and then were cultivated at a temperature of 26 • C and a relative humidity of 90% for a period of 5-7 days.The disease level of the rice plants was evaluated and captured through photodocumentation.The relative area of the lesions was calculated using ImageJ v1.46 software.

Pathogen Infection and Hormone Induction Treatment
SA, MeJA, or a conidial suspension was applied through spraying onto 2-week-old rice plants to investigate the expression of ATG genes at different time intervals.Total RNA was extracted from rice leaves using the Eastep ® Super Total RNA Extraction Kit (LS1040, Promega, Fitchburg, MA, USA).Subsequently, the RNA was reverse transcribed into cDNA using the 1st Strand cDNA Synthesis SuperMix for qPCR (11141ES60, Yeasen Biotechnology, Shanghai, China).The qRT-PCR analysis of rice OsATG genes was conducted using the qPCR SYBR Green Master Mix (11201ES08, Yeasen Biotechnology, Shanghai, China).The primer sequences can be found in Supplementary Table S4.

Subcellular Localization and Immune Response Analysis
The GFP (green fluorescent protein)-ATG and RFP (red fluorescent protein)-ATG fusion plasmids were constructed and then transformed into rice protoplasts.Following a transformation incubation lasting 12-24 h, the subcellular localization of ATG proteins was visualized using microscopy.Immune response experiments were conducted to evaluate callose deposition and reactive oxygen species (ROS) burst.These experiments were carried out following the methods described in a previously published article [48].

Analysis of Protein Interactions
The yeast two-hybrid screen was conducted to screen potential interacting proteins of the target protein.The BD-ATG plasmid was transformed with the plasmids of the AD-rice cDNA library into the yeast strain AH109.Subsequently, the resulting yeast transformants were subjected to amplification utilizing SF and SR primers, following which the amplified fragments were sent for sequencing.The obtained sequencing results were then aligned with sequences accessible on the NCBI database.Finally, the identified proteins underwent Gene Ontology (GO) analysis via the PANTHER website.

Discussion
M. oryzae is a pathogen that can infect various cereals [49].The economic impact of this fungus is significant, with annual losses estimated at around $66 billion, and the destruction of harvested grain could have fed 60 million people [50].While farmers can control rice blast through measures like crop rotation, proper water management, and judicious fertilizer use, breeding resistant cultivars is also effective.However, no cultivar is completely resistant to all races of M. oryzae, and resistant cultivars often lose their effectiveness within a few years as the fungus adapts [49,50].Therefore, it is crucial to identify candidate disease-resistant genes and understand the underlying mechanisms of immune defense responses to increase rice yield.
Unlike animals, plants must overcome or tolerate various environmental stresses such as high temperatures, freezing, drought, flooding, herbivory by animals and insects, and invasion by pathogens [51,52].Autophagy, a process that helps maintain cellular homeostasis, plays a significant role in helping plants cope with these stresses [53,54].Plant ATG genes have been found to be involved in pathogen infection responses.However, the roles of different plant ATGs in plant immunity can vary due to variations in nutritional types and evolutionary directions among pathogens, and in some cases, they may even have opposite effects [55].To investigate the role of rice OsATGs in response to M. oryzae infection, we conducted bioinformatic analysis, gene expression pattern analysis, and disease resistance analysis on OsATGs.Our study aims to provide a theoretical foundation for understanding the role of autophagy in plant immune processes.
In this study, we identified 37 OsATGs in rice and conducted a series of bioinformatic analyses.Our analyses revealed significant variations in the physicochemical properties of different OsATGs (Table 1) and variations in the number of exons (Figure 2a).However, the distribution of functional domains and conserved motifs exhibited similarities.For example, all OsATG8 genes contained the "ubl-ATG8" domain and motif 1, while all OsATG18 genes contained the "WD40" domain and motif 3 (Figures 2b and 3a).These analytical results show similarities to the analysis of ATG homologs in other species [11,30].
Plant hormones play a crucial role in the growth and development of plants.However, they also function as cell signaling molecules and play an important role in defending against pathogen invasion [56].Salicylic acid (SA) and jasmonic acid (JA) are key participants in plant immunity.They are able to enhance immune signal transduction and activate the expression of defense-related genes, thus promoting disease resistance [57][58][59].In this study, we found that 13 and 26 promoter regions of OsATGs contain cis-acting elements responsive to SA and JA, respectively.Among them, six OsATGs were found to respond to both SA and JA simultaneously (Figure 4b).In the subsequent SA and JA induction experiments, the results showed that five genes responded to SA and JA induction, and among them, four genes (OsATG6c, OsATG8a, OsATG9b, and OsATG13a) were able to simultaneously respond to SA and JA induction (Figure 6).These results suggest the involvement of rice OsATGs in plant immune responses mediated by SA and JA.
Studying the gene expression patterns of plants during the infection stage of pathogens is crucial for identifying important candidate resistance genes [60].Additionally, it allows for the rapid detection of the expression status of genes involved in plant immune processes [60].Investigating the gene expression changes of plant ATG genes during the infection stage can provide insights into the role of autophagy in plant immunity.For instance, studies on banana and cassava have shown that there are eight and twenty-five upregulated ATG genes, respectively, during pathogen infection [61,62].In our study, we observed diverse changes in the expression of OsATGs after 0, 24, and 48 h of M. oryzae infection.Specifically, seven OsATGs consistently showed upregulation, while ten OsATGs consistently showed downregulation (Figure 5a).During the infection stage of rice blast fungus, we observed significant changes in the gene expression of five OsATGs compared to 0 h (Figure 5d).These findings suggest that different OsATGs may play distinct roles in response to M. oryzae infection.
Autophagy genes can be categorized into four systems, including the ATG1-ATG13 complex, the ATG9-ATG2 complex, the PI3K-ATG6 complex, and the ATG8-ATG12 ubiquitination system.Each system has a distinct function in autophagy, including autophagy initiation, membrane supply for autophagosome formation, nucleation of autophagosomes, and membrane expansion of autophagosomes, respectively [1,63].To comprehensively elucidate the role of autophagy genes in rice immunity, we focused on studying one to two genes from each system, specifically OsATG13a, OsATG9a, OsATG9b, OsATG6b, OsATG6c, and OsATG8a.Resistance assays revealed that loss of function in OsATG6c, Os-ATG8a, OsATG9b, and OsATG13a led to decreased resistance against M. oryzae (Figure 5b,c).Furthermore, the osatg13a mutant showed reduced chitin-induced callose accumulation and ROS burst compared to the wild type (Figure 7).These findings suggest that autophagy in rice positively regulates the immune response to M. oryzae.
In this study, we have identified several key autophagy-related genes (OsATG6c, OsATG9b, OsATG8a, and OsATG13a) that contribute to rice resistance against M. oryzae infection.Interestingly, the role of ATG6 in plant immunity can vary depending on the specific pathogen.For instance, the loss of function of BrATG6 in cabbage (Brassica rapa) leads to increased susceptibility to Plasmodiophora brassicae, while the absence of TaATG6 in wheat enhances resistance against Blumeria graminis f. sp.tritici [64,65].Moreover, ATG9 and ATG8 have been found to play a positive regulatory role in plant immunity.The overexpression of NbATG9 in tobacco (Nicotiana benthamiana) significantly enhances autophagy activity, resulting in increased resistance against Phytophthora infestans, the causal agent of potato late blight [66].Similarly, in wheat, TaATG8j enhances resistance against Puccinia striiformis f.sp.tritici, the causal agent of stripe rust, by regulating cell death [67].However, there is limited information on the involvement of ATG13 in plant immunity.Therefore, in order to gain a better understanding of the potential mechanisms underlying its involvement in rice immunity, we conducted a screening for interacting proteins of OsATG13a.Our preliminary results suggest that OsRLK170579, OsKEG1, OsANR1, OsPP2A-4, OsVPE1, and OsMDH1 may be involved in plant immunity by interacting with OsATG13a.
Receptor-like kinases (RLKs) are essential protein kinases that are widely present in plants and play essential roles in growth, development, and immunity.Yin and Liu have developed RLKdb, a comprehensive RLKs database containing approximately 220,000 RLKs from 300 plant genomes [33].Among the RLKs included in the database, OsRLK170576 stands out as a potential key player in rice immunity.The E3 ligase KEEP ON GOING (KEG) is involved in ubiquitination processes.In Arabidopsis, KEG regulates the protein levels of MKK4 and MKK5 through ubiquitination, revealing a mechanism through which plants can finely regulate immune responses by modulating key MAPK cascade members [34].Proanthocyanidins (PAs), also known as condensed tannins, are significant defense-related phenolic compounds in mulberry trees (Morus alba), and their biosynthesis is linked to the expression of anthocyanidin reductase (ANR).Studies have shown that the heterologous expression of MnANR from mulberry in tobacco enhances tobacco's resistance to Botrytis cinerea [35].Serine/threonine protein phosphatases type 2A (PP2A) play crucial roles in growth, development, stress responses, and hormone signaling.In the potato (Solanum tuberosum), the overexpression of StPP2Ac2b, a member of PP2A, leads to plant senescence, resulting in decreased resistance to late blight disease [36].Vacuolar processing enzyme (VPE) is a cysteine protease involved in the maturation of vacuolar protein.In N. benthamiana, NbVPE plays a role in the hypersensitive response (HR) induced by elicitors, suggesting its involvement in immune processes triggered by elicitors [37].Malate dehydrogenase (MDH) is a critical metabolic enzyme involved in various plant development processes.In cassava (Manihot esculenta), MeMDH1 positively regulates cassava's resistance to Xanthomonas manihotis by modulating the accumulation of SA and the expression of pathogenesis-related protein 1 [38].Therefore, it is plausible that rice OsATG13a may participate in rice immune processes through interactions with OsRLK170576, OsKEG1, OsANR1, OsPP2A-4, OsVPE1, and OsMDH1.

Plants 2024 , 19 Figure 1 .
Figure 1.Phylogenetic and chromosomal localization analysis of OsATGs.(a) Phylogenetic analysis of OsATGs.The bootstrap values indicate the confidence of the branching.Clusters I-IV are represented by yellow, purple, green, and blue colors, respectively, while gray indicates individual groups.(b) Chromosomal localization analysis of OsATGs.The scale represents the distance on the chromosome, and the number of chromosomes is shown on the left side of each chromosome.Chromosomes are represented by gray boxes with green borders, while OsATG genes are displayed in red font.Rice has a total of 12 chromosomes, and OsATG genes are not distributed on Chr 9; hence, it is not shown here.

Figure 1 . 19 Figure 2
Figure 1.Phylogenetic and chromosomal localization analysis of OsATGs.(a) Phylogenetic analysis of OsATGs.The bootstrap values indicate the confidence of the branching.Clusters I-IV are represented by yellow, purple, green, and blue colors, respectively, while gray indicates individual groups.(b) Chromosomal localization analysis of OsATGs.The scale represents the distance on the chromosome, and the number of chromosomes is shown on the left side of each chromosome.Chromosomes are represented by gray boxes with green borders, while OsATG genes are displayed in red font.Rice has a total of 12 chromosomes, and OsATG genes are not distributed on Chr 9; hence, it is not shown here.Plants 2024, 13, x FOR PEER REVIEW 6 of 19

Figure 2 .
Figure 2. Gene structure and functional domain analysis of OsATGs.(a) Gene structure analysis of OsATG genes.The scale represents the gene length of OsATGs.The green box represents the untranslated region (UTR), the yellow box represents the coding sequence (CDS), and the unmarked regions represent introns.(b) Functional domain analysis of OsATG proteins.The scale represents the protein size of OsATGs, and the different colored boxes represent different functional domains.

Figure 2 .
Figure 2. Gene structure and functional domain analysis of OsATGs.(a) Gene structure analysis of OsATG genes.The scale represents the gene length of OsATGs.The green box represents the untranslated region (UTR), the yellow box represents the coding sequence (CDS), and the unmarked regions represent introns.(b) Functional domain analysis of OsATG proteins.The scale represents the protein size of OsATGs, and the different colored boxes represent different functional domains.

Figure 3 .
Figure 3. Motif analysis of OsATGs.(a) The distribution of 20 motifs in OsATGs.The scale represents the protein size of OsATGs, and the different colored boxes indicate the presence of different motifs.(b) The sequences of the 20 motifs in OsATGs.The height of each amino acid symbol is proportional to the conservation degree of the common sequence depicted by the 20 motifs.The shortest motif (motif 12) is only 21 amino acids and the longest motifs (motifs 1-5, 7, 8, 14, 19, and 20) are 50 amino acids.These sequence motifs are marked as created by MEME (motif-based sequence analysis tools).

Figure 4 .
Figure 4. Cis-acting elements analysis of OsATGs.(a) The distribution of all cis-acting elements in OsATGs.The scale represents the sequence length of 2kb upstream of the OsATG genes, and the boxes of different colors represent different cis-acting elements (CREs).There are a total of 24 CREs distributed in the promoter regions of 37 OsATGs.(b) The distribution of cis-acting elements related to plant hormones in OsATGs.The scale represents the sequence length of 2kb upstream of the OsATG genes, and the boxes of different colors represent different cis-acting elements.There are a total of 7 CREs related to plant hormones distributed in the promoter regions of 37 OsATGs.

Figure 4 .
Figure 4. Cis-acting elements analysis of OsATGs.(a) The distribution of all cis-acting elements in OsATGs.The scale represents the sequence length of 2kb upstream of the OsATG genes, and the boxes of different colors represent different cis-acting elements (CREs).There are a total of 24 CREs distributed in the promoter regions of 37 OsATGs.(b) The distribution of cis-acting elements related to plant hormones in OsATGs.The scale represents the sequence length of 2kb upstream of the OsATG genes, and the boxes of different colors represent different cis-acting elements.There are a total of 7 CREs related to plant hormones distributed in the promoter regions of 37 OsATGs.

Figure 5 .
Figure 5. Resistance of OsATG mutants to rice blast fungus.(a) Analyzing the expression pattern of OsATGs during M. oryzae infection at 0 h, 24 h, and 48 h using transcriptome data.The color gradient from green to red visually represents the regulation gradient from downregulation (green) to upregulation (red).(b) Resistance evaluation of OsATG mutants using spray inoculation.The 2-week-old rice seedlings were inoculated with M. oryzae conidial suspension of 1 × 10 4 mL −1 .The asterisk (*) indicates statistical significance at p < 0.05 (t-test).(c) Resistance evaluation of OsATG mutants using punch inoculation.The 8-weeks-old rice leaves were punched and injected with M. oryzae conidial suspension of 50 × 10 4 mL −1 .(d) Expression of selected OsATG genes during M. oryzae infection using qRT-PCR.The gene expression levels were calculated by the method of 2 −∆∆CT , where ∆T is calculated by subtracting the cycle threshold Ct value of the target gene from the Ct value of the Internal reference gene.Statistical significance levels were indicated by asterisks (* for p < 0.05,** for p < 0.01, *** for p < 0.001, **** for p < 0.0001, t-test).

Figure 7 .
Figure 7. Localization of OsATG13a and PTI responses of the osatg13a mutant.(a) Localization of OsATG13a in rice protoplast.Bar represents 10 µm.GFP can be localized throughout the entire cell, and GFP-OsATG13a has strong localization in the nucleus and small spots in the cytoplasm.(b) Co-localization of GFP-OsATG13a and RFP-OsATG16 in rice protoplast.Bar represents 10 µm.The yellow light at Merge is composed of a combination of green and red light, indicating that GFP-OsATG13a and RFP-OsATG16 share a common subcellular localization.(c) Callose deposition assay of ZH11 and osatg13a mutant.The bright white dots indicated by the red arrows are callosum, and the bar represents 100 µm.The more protein A there is, the stronger the plant's PTI response.(d) ROS burst assay of ZH11 and osatg13a mutant.The blue line represents water treatment in ZH11, the red line represents chitin treatment in ZH11, the green line represents water treatment in the osatg13a mutant, and the purple line represents chitin treatment in the osatg13a mutant.The higher the peak value, the stronger the plant's PTI response.

Figure 8 .
Figure 8. Screening and analysis of interacting proteins of OsATG13a.(a) PCR validation of yeast transformants.Maker represents maker III, and the sizes of the 7 bands in maker III are 4500, 3000, 2000, 1200, 800, 500, and 200 bp, respectively.The size range of bands used for sequencing is 500-2000 bp.(b) GO analysis of potential interacting proteins of OsATG13a.MF represents Molecular Function, CC represents Cellular Component, and BP represents Biological Process.Different colors of circles represent different categories, and the size of the circles represents the number of clusters.

Figure 8 .
Figure 8. Screening and analysis of interacting proteins of OsATG13a.(a) PCR validation of yeast transformants.Maker represents maker III, and the sizes of the 7 bands in maker III are 4500, 3000, 2000, 1200, 800, 500, and 200 bp, respectively.The size range of bands used for sequencing is 500-2000 bp.(b) GO analysis of potential interacting proteins of OsATG13a.MF represents Molecular Function, CC represents Cellular Component, and BP represents Biological Process.Different colors of circles represent different categories, and the size of the circles represents the number of clusters.

Table 1 .
Physicochemical properties and subcellular localization prediction of OsATGs.

Table 2 .
Possible interacting proteins that may participate in immunity.

Table 2 .
Possible interacting proteins that may participate in immunity.