Microbe-responsive proteomes during plant-microbe interactions between rice genotypes and the multifunctional Methylobacterium oryzae CBMB20

doi:10.21203/rs.3.rs-2372686/v1

Download PDF

Research Article

Microbe-responsive proteomes during plant-microbe interactions between rice genotypes and the multifunctional Methylobacterium oryzae CBMB20

https://doi.org/10.21203/rs.3.rs-2372686/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 05 May, 2023

Read the published version in Rice →

You are reading this latest preprint version

Background

Rice is colonized by plant growth promoting bacteria (PGPB) such as Methylobacterium leading to mutually beneficial plant-microbe interactions. As modulators of rice developmental process, Methylobacterium influences seed germination, growth, health, and development. However, little is known on the complex molecular responsive mechanisms modulating microbe-driven rice development. The application of proteomics to rice-microbe interactions helps us elucidate dynamic proteomic responses mediating this association.

Results

In this study, a total of 3908 proteins were detected across all treatments of which the non-inoculated IR29 and FL478 share up to 88% similar proteins. However, intrinsic differences appear in IR29 and FL478 as evident to the differentially abundant proteins (DAPs) and their associated gene ontology terms (GO). Successful colonization of M. oryzae CBMB20 in rice resulted to dynamic shifts in proteomes of both IR29 and FL478. The GO terms of DAPs for biological process in IR29 shifts in abundance from response to stimulus, cellular amino acid metabolic process, regulation of biological process and translation to cofactor metabolic process (6.31%), translation (5.41%) and photosynthesis (5.41%). FL478 showed a different shift from translation-related to response to stimulus (9%) and organic acid metabolic acid (8%). Both rice genotypes also showed a diversification of GO terms due to inoculation of M. oryzae CBMB20. Specific proteins such as peptidyl-prolyl cis-trans isomerase (A2WJU9), thiamine thiazole synthase (A2YM28), and alanine—tRNA ligase (B8B4H5) upregulated in IR29 and FL478 indicate key mechanisms of M. oryzae CBMB20 mediated plant growth promotion in rice.

Conclusions

Interaction of Methylobacterium oryzae CBMB20 to rice results to a dynamic, similar, and plant genotype-specific proteomic changes supporting associated growth and development. The multifaceted CBMB20 expands the gene ontology terms and increases the abundance of proteins associated to photosynthesis, diverse metabolic processes, protein synthesis and cell differentiation and fate potentially attributed to the growth and development of the host plant. The specific proteins and their functional relevance help us understand how CBMB20 mediate growth and development in their host under normal conditions and potentially link subsequent responses when the host plants are exposed to biotic and abiotic stresses.

Proteomics

LC-MS/MS

Plant growth promotion

Methylobacterium

Rice

Plants are continually associated with microorganisms throughout their lifecycle with their associations established at the seed level even before they begin to germinate. These microbes are major factors that drive growth, intermittent or continuous stress tolerance, and the overall plant productivity which works vice versa as plants also influence their associated microbial community and function (Chialva et al. 2022). Rice plants have been found to establish intricate relationships with beneficial microbes from the soil and their phyllosphere forming endophytic plant-microbe associations that lead to enhanced plant growth and development (Sessitch et al., 2012; Hardoim et al., 2011, 2012). In many cases, beneficial plant-microbe interactions also lead to enhanced plant fitness and protection under biotic and abiotic environmental stresses (Pieterse et al., 2014; Balmer et al., 2015). Studies on the interactions of rice with bacterial endophytes also reveal complex microbial communities (Sun et al., 2008; Hardoim et al., 2011; Hardoim et al., 2012; Walitang et al., 2018), the functional properties of these microbial communities as well as their interaction mechanisms with rice (Sessitch et al., 2012; Walitang et al., 2017).

Elucidating and understanding these complex plant-microbe interactions, through omics studies, provide potential avenues to improve rice productivity while supporting sustainable agriculture and environmental resilience. In addition, determinants of successful and beneficial plant-microbe interactions greatly depend on the host plant, the microbial nature, and the environmental conditions. The host genotype generally dictates the recruitment of microbial inhabitants and the plant response to endophytes are conditioned by the plant genotype (Rosenblueth and Martinez-Romero, 2006; Hardoim et al., 2011; Johnston-Monje and Raizada, 2011). On the other hand, competent bacterial endophytes also possess adaptations that allow long-term persistence particularly through proteins and metabolites that promote plant growth and health (Hardoim et al., 2008; Hardoim et al., 2015). In rice plants, these endophytes can transfer across generations through their seeds and majorly contribute to a plant’s disease resistance (Walitang et al., 2019; Matsumoto et al., 2021).

Among the rice associated microbial communities, the genus Methylobacterium is a multifaceted, PGPB microbial group. Methylobacterium spp. are potentially ubiquitous to all plants and are substantially involved in some plant biological processes (Holland and Polacco, 1994). The strain Methylobacterium oryzae CBMB20 also exemplifies this group. CBMB20 was isolated in the stem endosphere of Oryza sativa subsp. japonica cv. Nam-peong (Madhaiyan et al., 2004; Madhaiyan 2007a). From its initial detection as a potential diazotroph, CBMB20 was then used and proven to promote growth in multiple plant hosts including other Oryza sativa japonica cultivars (Lee et al., 2006; Madhaiyan et al., 2010; Roy Choudhury et al., 2022), Oryza sativa indica cultivars (Chatterjee et al., 2019), red pepper (Ryu et al., 2006; Madhaiyan et al., 2010; Lee et al., 2011), canola (Madhaiyan et al., 2007b), soybean (Subramanian et al., 2014), and tomato (Ryu et al., 2006; Indiragandhi et al., 2008; Madhaiyan et al., 2010; Yim et al., 2013; Yim et al., 2014; Chanratana et al., 2018; Chanratana 2019). Inoculated CBMB20 could persist in the rhizosphere and phyllosphere (Lee et al., 2011; Madhaiyan et al., 2010), the spermosphere (Chanratana et al., 2018), and endosphere (Lee et al., 2011; Yim et al., 2013; Yim et al., 2014).

M. oryzae CBMB20 ability to promote plant growth in multiple plant host could be attributed to its ability to produce indole acetic acid (IAA) and cytokinin (Lee et al., 2006; Ryu et al., 2006; Madhaiyan et al., 2007b). It is also involved with plant nutrient absorption and dynamics through thiosulfate oxidation (Anandham et al., 2007), potential nitrogenase, phosphatase and urease activity (Madhaiyan et al., 2004; Madhaiyan et al., 2010). One of its most crucial attributes is the production and activity of 1-aminocyclopropane-1-carboxylate (ACC) deaminase enzyme. This enzyme modulates plant stress ethylene levels by catabolic hydrolysis of ACC, enhancing stress tolerance and restoring continued plant growth (Madhaiyan et al., 2007b; Indiragandhi et al., 2008; Yim et al., 2013; Yim et al., 2014; Chanratana 2018; Chatterjee et al., 2019; Roy Choudhury et al., 2022). The whole genome sequencing of CBMB20 supports the multifaceted nature of this versatile PGPB. Compared to closely related Methylobacterium strains, CBMB20 has more genes encoding motility and signaling indicating its more efficient responses to environmental cues and phyllosphere/endosphere colonization. M. oryzae CBMB20 is also equipped with genes related to PGP including genes for producing auxin, cytokinin, ACC deaminase, PQQ, and vitamin B12, as well as those involved with nutrient mobilization such as genes encoding for phytase, C-P lyase system, sulfur-oxidation, urease, and their associated transporters (Kwak et al., 2014).

Many of the studies involving PGPB are conducted at the physiological and organismal level involving selected parameters. However, plant-microbe interaction especially with a multifaceted PGPB involves dynamic and systemwide physiological changes mediated by transcripts, proteomes and metabolites. Plant-microbe interaction is not a simple outcome of one-way interaction but rather multigenic/multilevel plant responses. Proteomic analyses in recent times have proved the beneficial role of endophytic bacteria in activation of stress responses and survival mechanism of Eichhornia (Priya et al. 2022). Therefore, proteome analysis through high throughput sequencing, quantification and identification involved in plant-microbe interactions can help us better understand the complex networks that mediate such interactions.

The current study evaluates proteins and proteomes of two rice genotypes, IR29 and FL478 and how their proteomes change due to inoculation of the multifaceted endophytic Methylobacterium oryzae CBMB20 identifying microbe-responsive proteins providing insights into the molecular mechanisms allowing PGPB to directly and indirectly promote plant growth. Upon successful identification, the proteome can provide valuable information towards understanding the complex and fine-tuned plant-microbe interactions in rice and will help link future plant responses such as cross-tolerance to different stress conditions mediated by M. oryzae CBMB20.

Bacterial strain and preparation of inoculum

Methylobacterium oryzae CBMB20 was used as an inoculum for this study. The bacterial strain was grown in ammonium mineral salt (AMS) medium with 0.5% sodium succinate as a carbon source grown until OD₆₀₀ ~ 0.8. The strain was harvested using 0.03 M MgSO₄. For confocal microscopy, a previously tagged M. oryzae CBMB20-gfp (Kim et al., 2013) was used as inoculant and grown and prepared in the same condition as the wild type except that the AMS medium was supplemented with 50 µg mL^− 1 kanamycin.

Plant Material, Growth Conditions And Bacterial Inoculation

Rice (Oryza sativa L. subsp. indica cv. IR29; cv. FL478) seeds were obtained from the Rural Development Administration (RDA), Republic of Korea. Rice seeds were surface sterilized (Chatterjee et al., 2019) and germination was induced by soaking them in distilled water then incubating them without light for 2 days. Bacterial inoculation and seedling transplantation were done according to Roy Choudhury et al. (2022). Germinated seeds were sown in nursery soil and grown in greenhouse conditions under natural illumination. Seedlings (7-day-old) were bacterized by dipping the roots in the inoculum suspension for 2 min, transplanted in seedling trays (individual hole diameters: 5 cm – outer diameter; 3 cm – inner diameter; 6 cm – depth) containing nursery soil and an additional 5 mL inoculum was added in the rhizosphere region. A solution of 0.03 M MgSO₄ was used for non-inoculated plants. The same set-up was made for rice plants inoculated with M. oryzae CBMB20-gfp to be used for confocal microscopy. The experiment was designed with four treatments namely, non-inoculated IR29 (IR29-NI), non-inoculated FL478 (FL478-NI), M. oryzae CBMB20 inoculated IR29 (IR29-I), and M. oryzae CBMB20 inoculated FL478 (FL478-I) and replications for each treatment.

Confocal Microscopy

Freshly harvested 14-day-old IR29 and FL478 plant samples (roots, shoots, and stem) were removed from at least three seedling plants inoculated with M. oryzae CBMB20-gfp, washed with sterile distilled water then blotted dry. Hand-cut transverse and cross sections of surface-sterilized roots, shoots, and stems were used to check the endophytic colonization by the inoculated strains. The samples were mounted using sterile glycerol under a coverslip. The samples were stored at 4°C for 24 h prior to confocal microscopy. Microscopic observations were performed using Leica TCS SP2 confocal system (Leica Microsystems Heidelberg GmbH, Manheim, Germany) equipped with an Arion laser (gfp: excitation, 488nm; emission filter BP500-530). Image acquisitions were carried out using a ×20 and ×50 objectives and processed with Zen software package (version 2.5.1227a).

Protein Extraction, Purification And Digestion

Seedlings (14-day-old) of IR29 and FL478 were harvested containing 30 plants per replicate. Fresh leaf samples (0.5 g) were finely ground with liquid nitrogen and TCA (trichloroacetic acid)/Acetone precipitation method for protein extraction was done according to Roy et al. (2016). Bradford method (1976) was used to quantify the extracted total protein content. The methanol-chloroform method (Komatsu et al., 2013) was used to purify 150 µg protein samples and were alkylated with iodoacetamide before digesting with trypsin.

Lc-ms/ms Analysis

LC-MS/MS analysis of digested protein samples was done using an LTQ Orbitrap mass spectrometer (Thermo Fischer, Bremen, Germany) coupled with 1100 nano-flow HPLC system (Agilent) equipped with a nano electrospray ion source. This two-column system consist of an interconnected (three-way tee connector) pre-column, waste line and the analytical column (C18 AQ, 3 µm, 100 µm x 15 cm, NanoLC, USA). Aliquots of the peptide samples (10 µL) were loaded with an autosampler into the C18 trap column (Accaim Pep Map 100, 75 µm x 2 cm, nano Viper C18, 3 µm, Thermo Fischer Scientific, USA). The mobile phases, A and B, were used containing 0 and 100% acetonitrile, respectively, each supplemented with 0.1% formic acid. The trap column desalted and concentrated the peptide samples at a flow rate of 10 µL min^− 1 for 10 min with buffer A followed by elution with buffer B with a 150 min, 0–50% linear gradient at a flow rate of 300 nL min^− 1. To avoid sample carryover, the column was washed with 10 column volumes of buffer B after elution followed by re-equilibration with buffer A. All MS/MS spectra were acquired for fragmentation of the five most abundant peaks in a data-dependent mode with 35% normalized collision energy. An electronspray voltage of 2.2 kV was used to enter the separate peptides into the mass spectrometer. The dynamic exclusion duration was 180 sec with exclusion mass width of 0.5 Da. A mass spectral range of 150–2000 m/z was used for acquiring MS spectra.

Protein Identification And Quantification

The obtained MS/MS spectra of proteins were searched in the universal Protein Resource Database (http://www.uniprot.org) using Mascot Daemon (Version 2.4.1, Matrix Science, London, UK). Criteria for identification of peptides were based on monoisotopic mass selection, precursor mass tolerance of ± 1.5 Da, fragment mass tolerance of ± 0.8 Da, two missed trypsin cleavage and fixed modification of carbamidomethyl cysteine. For each database search, the calculated threshold score based on the Mascot ion score threshold (0.5) was used for accepting individual spectrum. The false discovery rates (FDR) for peptides and proteins were calculated by Peptide Validator at 1.0% above identity threshold.

The label-free quantitation analysis using emPAI was performed according to Shinoda et al. (2010). Common contaminant and decoy matches were filtered from the protein identification list. For the determination of protein identification, at least two unique peptides per protein were required. Relative quantification was done for proteins detected in at least two technical replicates of three biological trials in each group. An arithmetic mean was used to obtain the average label-free quantification within each biological group and statistically evaluated through a two-sided t-test. The calculated p-value was corrected using false discovery rate-based multiple hypothesis testing using Perseus statistics software. All data were normalized using linear regression analysis.

Bioinformatics Analysis

Fold changes of the differentially abundant proteins (DAPs) were calculated by comparing the abundances among the treatment comparisons. Significantly more abundant and less abundant proteins were categorized using DAVID Bioinformatics Tools (https://david.ncifcrf.gov) based on biological processes, molecular functions, and cellular components. Various metabolic pathways were determined through the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/pathway.html).

Statistical Analysis And Visualization

The experiment was carried out in a completely randomized block design. Heatmaps were generated with the web-based tool clustvis (Metsalu and Vilo, 2015). Venn diagrams were visualized using the web-based tool Venny (Oliveros, 2015).

Systemic endophytic colonization of M. oryzae CBMB20

Root, stem and leaf samples of IR29 and FL478 were prepared after 7 days post inoculation for confocal microscopy. The CBMB20-gfp tagged cells could be easily distinguished as green fluorescing bacteria in most of the samples prepared even with high background fluorescence. CBMB20 could be seen to persist in the root rhizosphere occupying the root hair junction and on the surfaces of the root hairs (Fig. 1A, 1B). Endophytic colonization was successful for both IR29 (Fig. 1B-1D) and for FL478 (Fig. 1F-1H). Systemic endophytic colonization was observed as CBMB20 internally colonized the roots (Fig. 1B, 1F), stems (Fig. 1C, 1G), and leaves (1D, 1H). Phyllospheric colonization could also be observed as few cells were detected on the leaf surface of FL478 (Fig. 1H). Specific internal colonization could be observed in the intercellular regions (Fig. 1B, 1D, 1G, 1H) and xylem elements (1F).

Overall Proteomics Of Ir29 And Fl478

Through gel-free quantitative proteomics approach, a total of 3908 proteins were detected in rice plants across all the treatments at a 95% confidence level. A total of 1267, 1323, 821 and 1734 proteins are found IR29-NI, FL478-NI, IR29-I, and FL478-I, respectively (Fig. 2). The number of common proteins found in all treatments range from 41% − 87% relative to their individual total protein. However, the similarity between the non-inoculated IR29 (IR29-NI) and FL478 (FL478-NI) is around 84% – 88%. Differentially abundant proteins (DAPs) could be observed when comparing treatments. It was observed that the abundances of 28 and 52 proteins were significantly increased and decreased, respectively, in IR29-NI vs. FL478-NI treatment comparison. On the other hand, 54 and 6 proteins, 20 and 34 proteins, and 2 and 114 proteins were increased and decreased in IR29-NI vs. IR29-I, FL478-I vs. FL478-I, and IR29-I vs FL478-I treatment comparisons, respectively (Fig. 3, Additional file 2. Table S1).

Gene Ontology (Go) Terms Of Proteomes In Ir29 And Fl478 During The Early Seedling Development

The comparison of the IR29-NI and the FL478-NI could infer inherent phenotypic differences between the two contrasting rice genotypes in terms of their expressed proteomes during the rapid early seedling developmental stages. The GO enrichment-based cluster analysis of the more abundant and less abundant proteins was determined in terms of biological process (Fig. 4; Additional file 2. Table S2), cellular component (Fig. 5; Additional file 2. Table S2), and molecular function (Fig. 6; Additional file 2. Table S2). The majority of the DAPs that are more abundant in IR29 belonged to response to stimulus (9.09%), regulation of biological process (6.82%), translation (6.82%), and cellular amino acid metabolic process (6.82%) in the biological process category (Fig. 4). These DAPs are mainly associated to cytoplasm (25.71%), membrane (14.29%), plastid (11.42%) and the ribonucleoprotein complex (11.43%) (Fig. 5) carrying out the molecular functions for ion binding (19.57%) and nucleotide binding (14.13%) (Fig. 6, Additional file 4. Table S3). On the other hand, the majority of the DAPs that are more abundant in FL478 clustered in translation (11.43%) followed by less but equally abundant proteins in the biological process category (Fig. 4). These proteins are mainly localized in the plastids (26.92%) and membranes (15.38%) associated to molecular functions mainly transferase activity (16.67%), ion binding (11.9%) and structural molecule activity (11.9%) (Fig. 5, Fig. 6, Additional file 5. Table S4).

Gene ontology (GO) terms of proteomes in IR29 and FL478 during M. oryzae CBMB20 inoculation

Candidate DAPs in rice changing in abundance due to M. oryzae CBMB20 interactions could be inferred by comparing the IR29-NI vs. IR29-I and FL478-NI vs. FL478-I. Additional information could also be derived when comparing IR29-I and FL478-I (Additional file 6. Table S5, Additional file 7. Table S6, Additional file 6. Table S7). The GO enrichment-based cluster analysis for biological process (Fig. 7), cellular component (Fig. 8), and molecular function (Fig. 9) of significantly abundant DAPs show potential rice genotype individual responses, similarities, and differences when interacting with CBMB20. The GO terms of DAPs for biological process in IR29 shifts in abundance from response to stimulus, cellular amino acid metabolic process, regulation of biological process and translation to cofactor metabolic process (6.31%), translation (5.41%) and photosynthesis (5.41%). The rice genotype FL478 showed a different shift in protein abundance from translation related to response to stimulus (9%) and organic acid metabolic acid (8%) (Fig. 7). The cellular components to which these DAPs are associated also shifts from cytoplasm-related to plastid (26.92%) and membrane (11.53%) for IR29 and from plastid-associated to a more balanced plastid (18%), membrane (18%), and cytoplasm-associated (18%) proteins in FL478 (Fig. 8). These DAPs in IR29 and FL478 are mainly carrying the molecular function of ion binding, nucleotide binding, hydrolase activity, and oxidoreductase activity (Fig. 9). In general, rice-M. oryzae CBMB20 interactions lead to shifts in abundance of DAPs, quite distinct for both IR29 and FL478. It is also noteworthy to emphasize that there were expansion of GO terms of the biological process and molecular functions due to inoculation of CBMB20, although the relative abundances of proteins associated to these GO terms were generally much lower.

Proteomes responsive to M. oryzae CBMB20 inoculation in IR29 and FL478

The significantly abundant proteomes in IR29 and FL478 consisting of 60 and 54 DAPs, respectively, are associated with major functions and processes contributing to overall plant growth and development. Specific proteins associated to photosynthesis and metabolism were more prominently abundant in IR29 (Fig. 10) while specific proteins associated to protein folding and transport, membrane and transport, cell structure, and cell division, differentiation and fate were more distinctly abundant in FL478 (Fig. 10; Additional file 6. Table S5). Out of 54 significantly abundant DAPS in IR29, seven are mainly photosynthesis-related proteins including NADPH-protochlorophyllide oxidoreductase (A2XZO1), protein THYLAKOID FORMATION1 (A2YML2), ATP synthase sububit b, chloroplastic (P0C2Y9), photosystem I P700 chlorophyll a apoprotein (P0C3557), photosystem I iron-sulfur center (P0C360), protein RETICULATA-RELATED 1, chloroplastic-like (A2ZJL3), and thylakoid luminal 16.5 kDa protein, chloplastic (B8B267). These photosynthesis related proteins are mainly associated to the thylakoid membrane of the plastid. There are also significantly abundant DAPs in IR29 involved in numerous metabolic processes and protein synthesis. On the other hand, significantly abundant DAPs in FL478 are associated to metabolism and protein synthesis as well as cell structure, organization, differentiation and fate include outer envelope protein 80 (A2X208), oxalate- - CoA ligase (A2XZ41), V-type proton ATPase subunit C (B8AXG9), and mitochondrial import receptor subunit TOM20 (A2WYG9) (Fig. 10, Additional file 7. Table S6).

A few patterns could also be observed when assessing effects of CBMB20 on the proteomes of IR29 and FL478. More notably, there are similar proteins that were upregulated in both IR29 and FL478 upon CBMB20 inoculation. Enzymes peptidyl-prolyl cis-trans isomerase (A2WJU9), thiamine thiazole synthase (A2YM28), and alanine—tRNA ligase (B8B4H5) were all upregulated in CBMB20-inoculated IR29 and FL478 (Table 1; Additional File 1: Fig. S1). There are also proteins and enzymes with similar functional category downregulated due to CBMB20 inoculation. In FL478 observed in both FL478-NI vs FL478-I and IR29-I vs. FL478-I treatment comparisons, thioredoxin H-type (A2YIW7), glutathione transferase (A2Z9L8), enoyl reductase (B8B9S9), oxysterol-binding protein-related protein (A2XF220) and cytochrome b6 were lower in abundance (Table 2) associated to stress and defense and signaling. Although there are only six downregulated DAPs in IR29, the protein thioredoxin domain-containing protein, a stress-related protein was also similarly observed.

Table 1

Common upregulated DAPS between IR29 and FL478 due to CBMB20 inoculation.
Upregulated DAPs in IR29 due to inoculation of CBMB20	Upregulated DAPs in FL478 due to inoculation of CBMB20	Protein function
A2WJU9 Peptidyl-prolyl cis-trans isomerase	A2WJU9 Peptidyl-prolyl cis-trans isomerase; (Short = PPIase; EC = 5.2.1.8)	0006464: cellular protein modification process
A2YM28 Thiamine thiazole synthase, chloroplastic; (AltName: Thiazole biosynthetic enzyme)	A2YM28 Thiamine thiazole synthase, chloroplastic; (AltName: Full = Thiazole biosynthetic enzyme; EC = 2.4.2.60)	0050896: response to stimulus; 0006725: cellular aromatic compound metabolic process; 0006766: vitamin metabolic process; 0006790: sulfur compound metabolic process; 0006807: nitrogen compound metabolic process; 0017144: drug metabolic process; 0046483: heterocycle metabolic process
B8B4H5 Alanine–tRNA ligase, chloroplastic/mitochondrial precursor	B8B4H5 Alanine–tRNA ligase, chloroplastic/mitochondrial precursor; (EC = 6.1.1.7; AltName: Full = Alanyl-tRNA synthetase; Short = AlaRS)	0006412: translation; 0006520: cellular amino acid metabolic process; 0016070: RNA metabolic process

Table 2

Downregulated DAPS in FL478 due to *Methylobacterium oryzae* CBMB20 inoculation.
Downregulated DAPs in FL478 due to inoculation of CBMB20 (Protein Accession Number)	Downregulated DAPs in FL478 due to inoculation of CBMB20 (Protein Name)	Protein function
B8B9S9	Enoyl-[acyl-carrier-protein] reductase [NADH] 1, chloroplastic	0006082: organic acid metabolic process; 0044255: cellular lipid metabolic process
A2YIW7	Thioredoxin H-type; (Short = Trx-H; AltName: Full = Phloem sap 13 kDa protein 1)	0006810: transport; 0050896: response to stimulus
A2Z9L8	Glutathione transferase; (EC = 2.5.1.18)	0006518: peptide metabolic process; 0006790: sulfur compound metabolic process; 0051186: cofactor metabolic process; 0050896: response to stimulus
P0C316	Cytochrome b6	0015979: photosynthesis; 0022900: electron transport chain; 0045333: cellular respiration
A2XF22	Oxysterol-binding protein-related protein 3B	0008289: lipid binding

The current study was conducted to determine how bacterial inoculation with a multifunctional plant growth promoting M. oryzae CBMB20 affects the expression of proteins in rice plants, IR29 and FL478, and promotes plant growth and development during the rapid changes occurring during the early seedling development. We initially observed the endophytic colonization of CBMB20 in IR29 and FL478. To establish inherent phenotypic proteomic differences between IR29 and FL478, IR29-NI and FL478-NI were compared. To assess microbe-mediated proteomic changes due to CBMB20 inoculation, treatment comparison between IR29-NI vs. IR29-I and FL478-NI vs. FL478-I were analyzed. Additional information could also be derived in the inoculated plants by comparing IR29-I vs. FL478-I.

Inherent Phenotypic Similarities And Differences Between Ir29 And Fl478 Expressed Through Their Proteomes

The cultivars IR29 and FL478 are genotypically closely related rice cultivars where FL478 traces its parental lineage to IR29 and Pokkali of which IR29 is the high yielding salt-sensitive parent and Pokkali is the salt-tolerant low yielding parent (Walitang et al., 2019). The present study validates the close relatedness of the two rice genotypes as almost 88% of their phenotypically expressed proteins during their early seedling development are exactly similar. However, the level of expression of these proteins potentially differs between the two rice cultivars as we observed 28 DAPs in salt tolerant FL478 compared to 52 DAPs in the sensitive rice variety IR29. The remaining differences in expressed proteins may further lead to distinct differences in IR29 and FL478. The differences in certain biological functions and processes observed in this study corroborated the transcriptional and translational differences between the parent genotypes observed in an earlier study (Li et al., 2018).

Overall, a majority of the DAPs associated to FL478 and IR29 are related to metabolism, protein synthesis, stress and defense, carbohydrate and energy metabolism, membrane and transport. However, inherent differences between the two rice genotypes become apparent especially under salt stress conditions in which proteomes and transcripts points to variations in salt-stress adaptive defenses between the two rice cultivars.

Previous comparative studies under salt stress between the two rice genotypes showed that FL478 show greater abundance and upregulation of genes and proteins associated to superoxide dismutase, peroxidase genes, fiber protein, and inorganic pyrophosphatase with concurrent higher relative growth rate and lower Na⁺/K⁺ in the roots (Hosseini et al., 2015; Senadheera et al., 2012). Differences in biological process as well as molecular function were also observed between IR29 and FL478 through their transcriptional profile (Mirdar Mansuri et al., 2019) which corroborates the observed differences in terms of proteomics in the present study even under normal conditions. The observed differences between the two rice genotypes under normal growing conditions could be mainly attributed to the differences in gene expression profiles, both transcriptome and translatome, as seen in the comparison between IR29 and FL478’s paternal parent, Pokkali, showing distinct differences between the two even without salt stress (Li et al., 2018). Under normal conditions, Li et al. (2018) observed that the gene ontology terms overrepresented in Pokkali include regulation of metabolism, gene transcription, cellular carbohydrate metabolism, and DNA conformation while photosynthesis, response to stress, cell wall macromolecule metabolism and organization, transport and aminoglycan metabolism were observed for IR29. This pattern is quite similar to the proteomes of IR29 and FL478 especially after inoculation with CBMB20.

Changes in the functional proteomes gene ontology terms in IR29 and FL478 induced by inoculation of the multi-faceted Methylobacterium oryzae CBMB20

Methylobacterium oryzae CBMB20 has been well documented in enhancing plant growth under normal (Lee et al., 2006; Ryu et al., 2006; Madhaiyan et al., 2010) and stress conditions (Indiragandhi et al., 2008; Yim et al., 2013; Yim et al., 2014; Chanratana et al., 2018; Chatterjee et al., 2019). Common features of M. oryzae CBMB20 plant growth promotion usually include enhanced germination, germination rate, and seedling vigor index together with increased shoot and root length and increased biomass. However, there are potential differences in the responses across crop plants due to inoculation of CBMB20 (Ryu et al., 2006; Madhaiyan et al., 2010). In the current study, distinct differences in cultivar responses even at proteome level could be observed between IR29 and FL478. Interestingly, inoculation with CBMB20 expanded the gene ontology terms of the different proteins in both IR29 and FL478. The proteomes of IR29 and FL478 presented a much more diversified gene ontology terms upon inoculation with CBMB20. This supports the idea that inoculation with CBMB20 has extended the phenotype of the rice plants (Dawkins, 1999; Hardoim et al., 2015) mediated by the microbe-responsive proteomes.

Comparing the functional categories of the proteins associated to the CBMB20 inoculated IR29 and FL478 shows that there are more proteins related to photosynthesis, metabolism and protein degradation in IR29 while more proteins are associated to metabolism, stress and defense, cell structure, differentiation and fate, and protein folding and transport were observed in FL478. Roy Choudhury et al. (2022) showed that under normal growing conditions, inoculation with CBMB20 of a rice japonica cultivar increased the chlorophyll contents and carotenoid content of the leaves. In the present study, a protein directly involved with pigment synthesis (NADPH-protochlorophyllide oxidoreductase – A2XZ01) and chlorophyll-carotenoid formation was upregulated in IR29. Other photosynthesis-related proteins detected are mainly involved in the integrity and function of the chloroplast. The upregulated proteomes of IR29 and FL478 due to CBMB20 seem to reflect the enriched functional categories of transcriptomes and translatomes in IR29 and Pokkali (Li et al., 2019) and may point to a notion that CBMB20 inoculation reinforces these inherent differences in the two genotypes. Earlier, metabolomic and transcriptomic changes in Arabidobsis inoculated by Pseudomonas fluorescens SS101 has been established to be a strain-specific response (van de Mortel et al., 2012). However, our study showed that there are also plant genotype-specific proteomic responses occurring due to inoculation such as enrichment of photosynthesis-related proteins in IR29 and proteins related to cell differentiation in FL478. Although CBMB20 is an efficient plant growth promoter in multiple crop plants, the observed plant genotype-specific responses could explain differences in the effectiveness of bioinoculants and the need to investigate compatibility of crop and microbial inoculants such as those observed under field conditions (Sanjenbam et al., 2022).

Common microbe-responsive proteins modulated by Methylobacterium oryzae CBMB20

Although there is a difference in the overall response of IR29 and FL478 to CBMB20 inoculation, there are similar proteins upregulated indicating similar modes of microbe-mediated proteomic changes in both rice genotypes. There were three proteins both upregulated in IR29 and FL478 upon CBMB20 inoculation viz peptidyl-prolyl cis-trans isomerase (A2WJU9), thiamine thiazole synthase, chloroplastic (A2YM28), and alanine—tRNA ligase, chloroplastic / mitochondrial precursor (B8B4H5).

The enzyme peptidyl-prolyl cis-trans isomerase catalyze the isomerization between the cis and trans form of peptide bonds (Lin et al., 2019) particularly certain proline residues involved in protein folding (Blecher et al., 1996) and cellular protein modification process (GO: 006464). This enzyme is also regulated by various environmental stresses and could act as a molecular timer in diverse plant physiological and pathological processes (Blecher et al. 1996). Thiamine thiazole synthase is essential for thiamine biosynthesis especially targeted to chloroplasts influencing chloroplast development (Feng et al., 2019). However, the thiamine thiazole synthase detected in IR29 and FL478 is also related to response to stimulus (GO: 0050896) and different metabolic processes (GO: 0006727 – aromatic compound, GO: 0006777 – vitamin, GO: 0006790 – sulfur compound, GO: 0006807 – nitrogen compound, and GO: 0046483 – heterocycle metabolic process). In addition, based on gene ontology, alanine—tRNA ligase chloroplastic/mitochontrial precursor (B8B4H5), which attaches alanine to tRNA, is associated with translation (GO: 0006412), cellular amino acid metabolic process (GO: 0016070), and RNA metabolic process (GO: 0016070). The upregulation or enrichment of exactly similar proteins or proteomes with analogous functions in the two rice genotypes is potentially due to the same responses brought about by CBMB20 producing metabolites and proteins involved with plant growth promotion including hormones (Lee et al., 2006; Ryu et al., 2006; Madhaiyan et al., 2010) and improvement of plant nutrient contents (Anandham et al., 2007; Madhaiyan et al., 2010; Kwak et al., 2014).

Methylobacterium oryzae CBMB20 has been also observed to induce defense responses and was able to increase resistance to subsequent biotic stresses indirectly promoting growth through plant health (Indiraghandi et al., 2008; Yim et al., 2013; Yim et al., 2014). Colonization of non-native bacterial endophytes usually results in the activation of the plant’s defense responses and the colonizer has to either evade or modulate these responses in order to beneficially coexist with their host (Pieterse et al., 2014; Balmer et al., 2015). Several defense-related proteins have been modulated after rice inoculation with CBMB20. In FL478, thioredoxin H-type (A2YIW7) and glutathione transferase (A2Z9L8) were repressed similar to the thioredoxin domain-containing protein (A2YL83) observed in IR29. These proteins are important for regulating redox reactions and redox-mediated defense signaling (Yoo et al., 2011; Hosseini et al., 2015; Chi et al., 2019). These stress responsive proteins are potentially regulated indirectly though reduction of stress ethylene (Indiraghandi et al., 2008; Yim et al., 2013; Yim et al., 2014). These results show that CBMB20 has the capacity to modulate defense responses in rice and different from those observed in pathogenic bacteria (Pieterse et al., 2014; Balmer et al., 2015) where their presence lead to hypersensitive response or induction of systemic acquired resistance aimed at eliminating the invading pathogens. The modulation of stress responses by CBMB20 contributes to the indirect mechanism of plant growth promotion as CBMB20 inoculation potentially results to tolerance enhancement when rice plant is exposed to biotic and abiotic stress.

The interaction of rice and the plant growth promoting bioinoculant, Methylobacterium oryzae CBMB20 leads to dynamic proteome responses potentially resulting to beneficial plant-microbe interactions. This study showed that the genotypically related rice cultivars IR29 and FL478 phenotypically express around 90% similar proteins during their early seedling developmental stages. There are common responsive proteins upon CBMB20 inoculation indicating similar mechanisms of plant growth promotion in IR29 and FL478, associated to activation of vitamin biosynthesis related process and protein synthesis related process. However, there are distinct responses of IR29 and FL478 upon CBMB20 inoculation including enhancement of photosynthesis in IR29 and increased proteins related to cell structure, cell differentiation and fate, and protein folding and transport in FL478. M. oryzae CBMB20 inoculation also regulated stress and defense-related proteins resulting to modulation of defense responses in rice upon bacterial inoculation. It would be interesting to see how responses in rice genotypes are linked to subsequent changes mediated by CBMB20 when rice hosts are exposed to biotic and abiotic stresses. In addition, integrated analysis of transcriptomes, proteomes and metabolomes is crucial in elucidating the intricately complex plant-microbe associations.

Acknowledgements

The main author would like to thank the Romblon State University President, Dr. Meriam P. Catajay-Mani for her support on the invited Post-Doctoral Fellowship of D.I.W. We would also acknowledge the revision done by Dr. Krishnamoorthy Rangasamy.

Author Contributions

Conceptualization, D.I.W. and T.S.; methodology, D.I.W. and A.R.C.; validation, D.I.W., A.R.C. and K.C.; formal analysis, D.I.W., K.C., S.H.Y.; investigation, D.I.W.; resources, T.S., Y.L.; data curation, D.IW., G.C., B.J., and A.R.J; writing—original draft preparation, D.I.W.; writing—review and editing, D.I.W., A.R.C., and P.S.; visualization, D.I.W.; supervision, T.S., Y.L.; project administration, T.S. and Y.L.; project support, G.C., B.J., A.R.J; funding acquisition, T.S., Y.L. and A.R.C. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Basic Science Research Program, National Research Foundation of Korea (NRF), Ministry of Education, Science and Technology [2021R1A2C1006608], Republic of Korea.

Availability of Data and Materials

The datasets supporting the conclusions of this article are included within the article and its additional files.

Ethics Approval and Consent to Participate

Not applicable

Competing Interests

The authors declare that they have no competing interests.

Anandham R, Indiragandhi P, Madhaiyan M, Kim K, Yim W, Saravanan VS, Chung J, Sa T (2007) Thiosulfate oxidation and mixotrophic growth of Methylobacterium oryzae. Can J Microbiol 53(7):869-76. https://doi.org/10.1139/W07-057
Balmer A, Pastor V, Gamir J, Flors V, Mauch-Mani B (2015) The ‘prime-ome’: towards a holistic approach to priming. Trends Plant Scie 20(7):443-52. https://doi.org/10.1016/j.tplants.2015.04.002
Blecher O, Erel N, Callebaut I, Aviezer K, Breiman A (1996) A novel plant peptidyl-prolyl-cis-trans-isomerase (PPIase): cDNA cloning, structural analysis, enzymatic activity and expression. Plant Mol Biol 32(3):493-504.
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1-2), pp.248-254. https://doi.org/10.1016/0003-2697(76)90527-3
Chanratana M, Han GH, Melvin Joe M, Roy Choudhury A, Sundaram S, Halim MA, Sa T (2018) Evaluation of chitosan and alginate immobilized Methylobacterium oryzae CBMB20 on tomato plant growth. Arch Agron Soil Sci 64(11):1489-502. https://doi.org/10.1080/03650340.2018.1440390
Chanratana M, Joe MM, Roy Choudhury A, Anandham R, Krishnamoorthy R, Kim K, Jeon S, Choi J, Choi J, Sa T (2019) Physiological response of tomato plant to chitosan-immobilized aggregated Methylobacterium oryzae CBMB20 inoculation under salinity stress. 3 Biotech (11):1-3. https://doi.org/10.1007/s13205-019-1923-1
Chatterjee P, Kanagendran A, Samaddar S, Pazouki L, Sa TM, Niinemets Ü (2019) Methylobacterium oryzae CBMB20 influences photosynthetic traits, volatile emission and ethylene metabolism in Oryza sativa genotypes grown in salt stress conditions. Planta 249(6):1903-19. https://doi.org/10.1007/s00425-019-03139-w
Chi YH, Koo SS, Oh HT, Lee ES, Park JH, Phan KA, Wi SD, Bae SB, Paeng SK, Chae HB, Kang CH (2019) The physiological functions of universal stress proteins and their molecular mechanism to protect plants from environmental stresses. Front Plant Sci 10:750.
Chialva M, Lanfranco L, Bonfante P (2022) The plant microbiota: composition, functions, and engineering. Curr Opin Biotechnol 73:135-42. https://doi.org/10.1016/j.copbio.2021.07.003
Dawkins R, Dennett D. 1999. The extended phenotype: the long reach of the gene. Oxford University Press, Oxford, United Kingdom.
Feng X, Yang S, Tang K, Zhang Y, Leng J, Ma J, Wang Q, Feng X (2019) GmPGL1, a thiamine thiazole synthase, is required for the biosynthesis of thiamine in soybean. Front Plant Sci 10:1546. https://doi.org/10.3389/fpls.2019.01546
Hardoim PR, Andreote FD, Reinhold-Hurek B, Sessitsch A, van Overbeek LS, van Elsas JD (2011) Rice root-associated bacteria: insights into community structures across 10 cultivars. FEMS Microbiol Ecol 77(1):154-64. https://doi.org/10.1111/j.1574-6941.2011.01092.x
Hardoim PR, Van Overbeek LS, Berg G, Pirttilä AM, Compant S, Campisano A, Döring M, Sessitsch A (2015) The hidden world within plants: ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiol Mol Biol Rev 79(3):293-320. https://doi.org/10.1128/MMBR.00050-14
Hardoim PR, Hardoim CC, Van Overbeek LS, Van Elsas JD (2012) Dynamics of seed-borne rice endophytes on early plant growth stages. PloS One 7(2):e30438. https://doi.org/10.1371/journal.pone.0030438
Hardoim PR, Van Overbeek LS, van Elsas JD (2008) Properties of bacterial endophytes and their proposed role in plant growth. Trends Microbiol 16(10):463-71. https://doi.org/10.1016/j.tim.2008.07.008
Holland MA, Polacco JC (1994) PPFMs and other covert contaminants: is there more to plant physiology than just plant? Annu Rev Plant Biol 45(1):197-209.
Hosseini SA, Gharechahi J, Heidari M, Koobaz P, Abdollahi S, Mirzaei M, Nakhoda B, Salekdeh GH (2015) Comparative proteomic and physiological haracterization of two closely related rice genotypes with contrasting responses to salt stress. Funct Plant Biol 42(6):527-42. https://doi.org/10.1071/FP14274
Indiragandhi P, Anandham R, Kim K, Yim W, Madhaiyan M, Sa T (2008) Induction of defense responses in tomato against Pseudomonas syringae pv. Tomato by regulating the stress ethylene level with Methylobacterium oryzae CBMB20 containing 1-aminocyclopropane-1-carboxylate deaminase. World J Microbiol Biotechnol 24(7):1037-45. https://doi.org/10.1007/s11274-007-9572-7
Johnston-Monje D, Raizada MN (2011) Conservation and diversity of seed associated endophytes in Zea across boundaries of evolution, ethnography and ecology. Plos One 6(6):e20396. https://doi.org/10.1371/journal.pone.0020396
Kwak MJ, Jeong H, Madhaiyan M, Lee Y, Sa TM, Oh TK, Kim JF (2014) Genome information of Methylobacterium oryzae, a plant-probiotic methylotroph in the phyllosphere. PloS One. 9(9):e106704. https://doi.org/10.1371/journal.pone.0106704
Komatsu S, Han C, Nanjo Y, Altaf-Un-Nahar M, Wang K, He D, Yang P (2013) Label-free quantitative proteomic analysis of abscisic acid effect in early-stage soybean under flooding. J Proteome Res Nov 1;12(11):4769-84. https://doi.org/10.1021/pr4001898
Lee HS, Madhaiyan M, Kim CW, Choi SJ, Chung KY, Sa TM (2006) Physiological enhancement of early growth of rice seedlings (Oryza sativa L.) by production of phytohormone of N₂-fixing methylotrophic isolates. Biol Fertil Soils. 42(5):402-8. https://doi.org/10.1007/s00374-006-0083-8
Lee MK, Chauhan PS, Yim WJ, Lee GJ, Kim YS, Park KW, Sa TM. (2011) Foliar colonization and growth promotion of red pepper (Capsicum annuum L.) by Methylobacterium oryzae CBMB20. J Appl Biol Chem 54(2):120-5.
Li YF, Zheng Y, Vemireddy LR, Panda SK, Jose S, Ranjan A, Panda P, Govindan G, Cui J, Wei K, Yaish MW (2018) Comparative transcriptome and translatome analysis in contrasting rice genotypes reveals differential mRNA translation in salt-tolerant Pokkali under salt stress. BMC genom 19(10):95-113. https://doi.org/10.1186/s12864-018-5279-4
Lin W, Bonin M, Boden A, Wieduwild R, Murawala P, Wermke M, Andrade H, Bornhäuser M, Zhang Y (2019) Peptidyl prolyl cis/trans isomerase activity on the cell surface correlates with extracellular matrix development. Commun Biol 2(1):1-1.
Madhaiyan M, Park MS, Lee HS, Kim CW, Lee KH, Seshadri S, Sa TM (2004) Phenotypic characterization of methylotrophic N₂-fixing bacteria isolated from rice (Oryza sativa L.). Korean J Soil Sci Fert 37(1):46-53.
Madhaiyan M, Kim BY, Poonguzhali S, Kwon SW, Song MH, Ryu JH, Go SJ, Koo BS, Sa TM (2007a) Methylobacterium oryzae sp. Nov., an aerobic, pink-pigmented, facultatively methylotrophic, 1-aminocyclopropane-1-carboxylate deaminase-producing bacterium isolated from rice. Int J Syst Evol Microbiol 57(2):326-31. https://doi.org/10.1099/ijs.0.64603-0
Madhaiyan M, Poonguzhali S, Sa T (2007b) Characterization of 1-aminocyclopropane-1-carboxylate (ACC) deaminase containing Methylobacterium oryzae and interactions with auxins and ACC regulation of ethylene in canola (Brassica campestris). Planta 226(4):867-76. https://doi.org/10.1007/s00425-007-0532-0
Madhaiyan M, Poonguzhali S, Kang BG, Lee YJ, Chung JB, Sa TM (2010) Effect of co-inoculation of methylotrophic Methylobacterium oryzae with Azospirillum brasilense and Burkholderia pyrrocinia on the growth and nutrient uptake of tomato, red pepper and rice. Plant and soil 328(1):71-82.
Madhaiyan M, Poonguzhali S, Sa T (2007) Characterization of 1-aminocyclopropane-1-carboxylate (ACC) deaminase containing Methylobacterium oryzae and interactions with auxins and ACC regulation of ethylene in canola (Brassica campestris). Planta 226(4):867-76.
Mirdar Mansuri R, Shobbar ZS, Babaeian Jelodar N, Ghaffari MR, Nematzadeh GA, Asari S (2019) Dissecting molecular mechanisms underlying salt tolerance in rice: a comparative transcriptional profiling of the contrasting genotypes. Rice 12(1):1-3. https://doi.org/10.1186/s12284-019-0273-2
Matsumoto H, Fan X, Wang Y, Kusstatscher P, Duan J, Wu S, Chen S, Qiao K, Wang Y, Ma B, Zhu G. (2021) Bacterial seed endophyte shapes disease resistance in rice. Nature plants 7(1):60-72. https://doi.org/10.1038/s41477-020-00826-5
Metsalu T, Vilo J (2015) ClustVis: a web tool for visualizing clustering of multivariate data using Principal Component Analysis and heatmap. Nucleic acids research. 43(W1):W566-70. https://doi.org/10.1093/nar/gkv468
Oliveros, J.C. (2007-2015) Venny. An interactive tool for comparing lists with Venn’s diagrams. https://bioinfogp.cnb.csic.es/tools/venny/index.html
Pieterse CM, Zamioudis C, Berendsen RL, Weller DM, Van Wees SC, Bakker PA (2014) Induced systemic resistance by beneficial microbes. Annu Rev Phytopathol 52:347-75. https://doi.org/10.1146/annurev-phyto-082712-102340
Priya P, Aneesh B, Sivakumar KC, Harikrishnan K (2022) Comparative proteomic analysis of saline tolerant, phosphate solubilizing endophytic Pantoea sp., and Pseudomonas sp. Isolated from Eichhornia rhizosphere. Microbiol Res 265:127217. https://doi.org/10.1016/j.micres.2022.127217
Rosenblueth M, Martínez-Romero E (2006) Bacterial endophytes and their interactions with hosts. Mol Plant-Microbe Interact 19(8):827-37. https://doi.org/10.1094/MPMI-19-0827
Roy Choudhury A, Roy SK, Trivedi P, Choi J, Cho K, Yun SH, Walitang DI, Park JH, Kim K, Sa T (2022) Label‐free proteomics approach reveals candidate proteins in rice (Oryza sativa L.) important for ACC deaminase producing bacteria‐mediated tolerance against salt stress. Environ Microbiol. 24(8): 3612-3624. https://doi.org/10.1111/1462-2920.15937
Roy SK, Kwon SJ, Cho SW, Kamal AH, Kim SW, Sarker K, Oh MW, Lee MS, Chung KY, Xin Z, Woo SH (2016) Leaf proteome characterization in the context of physiological and morphological changes in response to copper stress in sorghum. Biometals 29(3):495-513. https://doi.org/10.1007/s10534-016-9932-6
Ryu JH, Madhaiyan M, Poonguzhali S, Yim WJ, Indiragandhi P, Kim KA, Anandham R, Yun JC, Kim KH, Sa T (2006) Plant growth substances produced by Methylobacterium spp. and their effect on tomato (Lycopersicon esculentum L.) and red pepper (Capsicum annuum L.) growth. J Microbiol Biotechnol16(10):1622-8.
Sanjenbam P, Shivaprasad PV, Agashe D (2022) Impact of phyllosphere Methylobacterium on host rice landraces. Microbiol Spect. (10)4. https://doi.org/10.1101/2022.02.07.479485
Senadheera P, Tirimanne S, Maathuis FJ (2012) Long term salinity stress reveals variety specific differences in root oxidative stress response. Rice Sci 19(1):36-43. https://doi.org/10.1016/S1672-6308(12)60018-3
Sessitsch AN, Hardoim PA, Döring J, Weilharter A, Krause AN, Woyke TA, Mitter B, Hauberg-Lotte L, Friedrich F, Rahalkar M, Hurek T (2012) Functional characteristics of an endophyte community colonizing rice roots as revealed by metagenomic analysis. Mol Plant-Microbe Interact 25(1):28-36. https://doi.org/10.1094/MPMI-08-11-0204
Shinoda K, Tomita M, Ishihama Y (2010) emPAI Calc—for the estimation of protein abundance from large-scale identification data by liquid chromatography-tandem mass spectrometry. Bioinformatics. 26(4):576-7. https://doi.org/10.1093/bioinformatics/btp700
Subramanian P, Kim K, Krishnamoorthy R, Sundaram S, Sa T (2015) Endophytic bacteria improve nodule function and plant nitrogen in soybean on co-inoculation with Bradyrhizobium japonicum MN110. Plant Growth Regul 76(3):327-32.
Sun L, Qiu F, Zhang X, Dai X, Dong X, Song W (2008) Endophytic bacterial diversity in rice (Oryza sativa L.) roots estimated by 16S rDNA sequence analysis. Microbial ecology. 55(3):415-24. https://doi.org/10.1007/s00248-007-9287-1
van de Mortel JE, de Vos RC, Dekkers E, Pineda A, Guillod L, Bouwmeester K, van Loon JJ, Dicke M, Raaijmakers JM (2012) Metabolic and transcriptomic changes induced in Arabidopsis by the rhizobacterium Pseudomonas fluorescens SS101. Plant Physiol 160(4):2173-88. https://doi.org/10.1104/pp.112.207324
Walitang DI, Kim CG, Kim K, Kang Y, Kim YK, Sa T (2018) The influence of host genotype and salt stress on the seed endophytic community of salt-sensitive and salt-tolerant rice cultivars. BMC Plant Biol 18(1):1-6. https://doi.org/10.1186/s12870-018-1261-1
Walitang DI, Kim K, Madhaiyan M, Kim YK, Kang Y, Sa T (2017) Characterizing endophytic competence and plant growth promotion of bacterial endophytes inhabiting the seed endosphere of Rice. BMC Microbiol 17(1):1-3. https://doi.org/10.1186/s12866-017-1117-0
Walitang DI, Kim CG, Jeon S, Kang Y, Sa T (2019) Conservation and transmission of seed bacterial endophytes across generations following crossbreeding and repeated inbreeding of rice at different geographic locations. Microbiology Open. 8(3):e00662. https://doi.org/10.1002/mbo3.662
Yim W, Seshadri S, Kim K, Lee G, Sa T (2013) Ethylene emission and PR protein synthesis in ACC deaminase producing Methylobacterium spp. inoculated tomato plants (Lycopersicon esculentum Mill.) challenged with Ralstonia solanacearum under greenhouse conditions. Plant Physiol Biochem 67:95-104.
Yim WJ, Kim KY, Lee YW, Sundaram SP, Lee Y, Sa TM (2014) Real time expression of ACC oxidase and PR-protein genes mediated by Methylobacterium spp. in tomato plants challenged with Xanthomonas campestris pv. Vesicatoria. J Plant Physiol 171(12):1064-75. https://doi.org/10.1016/j.jplph.2014.03.009
Yoo KS, Ok SH, Jeong BC, Jung KW, Cui MH, Hyoung S, Lee MR, Song HK, Shin JS (2011) Single cystathionine β-synthase domain–containing proteins modulate development by regulating the thioredoxin system in Arabidopsis. The Plant Cell 23(10):3577-94. https://doi.org/10.1105/tpc.111.089847

No competing interests reported.

Additionalfile1.Fig.S1.png
Additional file 1. Fig. S1. Venn diagrams of common and unique upregulated (A) and downregulated (B) DAPs due to Methylobacterium oryzae CBMB20 inoculation observed in different treatment comparisons.
Additionalfile2.TableS1.Listofsignificantdifferentiallyabundantproteinsamongtreatmentcomparison.xlsx
Additional file 2. Table S1. List of significant differentially abundant proteins among treatment comparison.
Additionalfile3.TableS2.GeneontologytermsofallDAPsinIR29NIFL478NI.xlsx
Additional file 3. Table S2. Gene ontology terms of all DAPs in IR29-NI vs FL478-NI.
Additionalfile4.TableS3.GeneontologytermsofmoreabundantDAPsinIR29NI.xlsx
Additional file 4. Table S3. Gene ontology terms of more abundant DAPs in IR29-NI.
Additionalfile5.TableS4.GeneontologytermsofmoreabundantDAPsinFL478NI.xlsx
Additional file 5. Table S4. Gene ontology terms of more abundant DAPs in FL478-NI.
Additionalfile6.TableS5.GeneontologytermsofsignificantDAPsinIR29NIIR29Id1.xlsx
Additional file 6. Table S5. Gene ontology terms of significant DAPS in IR29-NI vs. IR29-I.
Additionalfile7.TableS6.GeneontologytermsofsignificantDAPsinFL478NIFL478I.xlsx
Additional file 7. Table S6. Gene ontology terms of significant DAPs in FL478-NI vs. FL478-I.
Additionalfile8.TableS7.GeneontologytermsofsignificantDAPSinIR29IFL478I.xlsx
Additional file 8. Table S7. Gene ontology terms of significant DAPs in IR29-I vs. FL478-I.

Download PDF

Journal Publication

published 05 May, 2023

Read the published version in Rice →

Editorial decision: Major revision
12 Mar, 2023
Reviews received at journal
03 Jan, 2023
Reviewers agreed at journal
29 Dec, 2022
Reviewers agreed at journal
27 Dec, 2022
Reviewers invited by journal
16 Dec, 2022
Editor assigned by journal
13 Dec, 2022
Submission checks completed at journal
13 Dec, 2022
First submitted to journal
13 Dec, 2022

You are reading this latest preprint version

Microbe-responsive proteomes during plant-microbe interactions between rice genotypes and the multifunctional Methylobacterium oryzae CBMB20

Status:

Journal Publication

Version 1

Abstract

Background

Results

Conclusions

Figures

Background

Materials And Methods

Bacterial strain and preparation of inoculum

Plant Material, Growth Conditions And Bacterial Inoculation

Confocal Microscopy

Protein Extraction, Purification And Digestion

Lc-ms/ms Analysis

Protein Identification And Quantification

Bioinformatics Analysis

Statistical Analysis And Visualization

Results

Overall Proteomics Of Ir29 And Fl478

Gene Ontology (Go) Terms Of Proteomes In Ir29 And Fl478 During The Early Seedling Development

Discussion

Inherent Phenotypic Similarities And Differences Between Ir29 And Fl478 Expressed Through Their Proteomes

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1