Microbe-Responsive Proteomes During Plant-Microbe Interactions Between Rice Genotypes and the Multifunctional Methylobacterium oryzae CBMB20.

BACKGROUND: Rice is colonized by plant growth promoting bacteria such as Methylobacterium leading to mutually beneficial plant-microbe interactions. As modulators of the rice developmental process, Methylobacterium influences seed germination, growth, health, and development. However, little is known about 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 in 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 the 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 in 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 with 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.

Inoculation of ACC deaminase-producing endophytic bacteria down-regulates ethylene-induced pathogenesis-related signaling in red pepper (Capsicum annuum L.) under salt stress.

Pathogenesis-related (PR) signaling plays multiple roles in plant development under abiotic and biotic stress conditions and is regulated by a plethora of plant physiological as well as external factors. Here, our study was conducted to evaluate the role of an ACC deaminase-producing endophytic bacteria in regulating ethylene-induced PR signaling in red pepper plants under salt stress. We also evaluated the efficiency of the bacteria in down-regulating the PR signaling for efficient colonization and persistence in the plant endosphere. We used a characteristic endophyte, Methylobacterium oryzae CBMB20 and its ACC deaminase knockdown mutant (acdS- ). The wild-type M. oryzae CBMB20 was able to decrease ethylene emission by 23% compared to the noninoculated and acdS- M. oryzae CBMB20 inoculated plants under salt stress. The increase in ethylene emission resulted in enhanced hydrogen peroxide concentration, phenylalanine ammonia-lyase activity, ß-1,3 glucanase activity, and expression profiles of WRKY, CaPR1, and CaPTI1 genes that are typical salt stress and PR signaling factors. Furthermore, the inoculation of both the bacterial strains had shown induction of PR signaling under normal conditions during the initial inoculation period. However, wild-type M. oryzae CBMB20 was able to down-regulate the ethylene-induced PR signaling under salt stress and enhance plant growth and stress tolerance. Collectively, ACC deaminase-producing endophytic bacteria down-regulate the salt stress-mediated PR signaling in plants by regulating the stress ethylene emission levels and this suggests a new paradigm in efficient colonization and persistence of ACC deaminase-producing endophytic bacteria for better plant growth and productivity.

Development of ACCd producer A. brasilense mutant and the effect of inoculation on red pepper plants.

Azospirillum is a plant-growth-promoting bacterium capable of colonizing and promoting growth in numerous crops of agronomic and horticultural significance. The objective of the present study is to develop Azospirillum brasilense CW903-acdS flocculating cells and to test their performance in promoting the growth of red pepper plants grown under salt stress. The flocculating CW903-acdS recorded 12.6, 37.3 and 91.6% higher ACCd activity at 50, 100 and 150 mM NaCl concentrations, respectively, compared to non-flocculating (normal) CW903-acdS cells. The flocculating CW903-acdS recorded 29.8 and 24.5% higher specific growth rates compared to non-flocculating CW903-acdS cells at 100 and 150 mM NaCl concentration, respectively. The flocculating CW903-acdS recorded 29.7 and 24.5% higher production of IAA-like molecule compared to the non-flocculating CW903-acdS at 100 and 150 mM NaCl concentration, respectively. Similarly, 27.5 and 25.7% higher ARA activity was observed with the flocculating CW903-acdS compared to the non-flocculating CW903-acdS type cells at 100 and 150 mM NaCl concentration, respectively. In the pot culture experiment at 50 and 100 mM NaCl concentration, CW903-acdS inoculated pepper plants recorded 9.4 and 4.7% less ethylene emission, when compared to plants inoculated with non-flocculating CW903-acdS cells. At 100 mM NaCl concentration, plants inoculated with flocculating CW903-acdS recorded 27.5% higher dry weight compared to plants inoculated with non-flocculating CW903-acdS cells. This study implied the significance of flocculating CW903-acdS with better stress amelioration and plant growth promotion in red pepper plants grown under salt-affected conditions due to the positive influence of ACCd activity.

Label-free proteomics approach reveals candidate proteins in rice (Oryza sativa L.) important for ACC deaminase producing bacteria-mediated tolerance against salt stress.

The omics-based studies are important for identifying characteristic proteins in plants to elucidate the mechanism of ACC deaminase producing bacteria-mediated salt tolerance. This study evaluates the changes in the proteome of rice inoculated with ACC deaminase producing bacteria under salt-stress conditions. Salt stress resulted in a significant decrease in photosynthetic pigments, whereas inoculation of Methylobacterium oryzae CBMB20 had significantly increased pigment contents under normal and salt-stress conditions. A total of 76, 51 and 33 differentially abundant proteins (DAPs) were identified in non-inoculated salt-stressed plants, bacteria-inoculated plants under normal and salt stress conditions respectively. The abundances of proteins responsible for ethylene emission and programmed cell death were increased, and that of photosynthesis-related proteins were decreased in non-inoculated plants under salt stress. However, bacteria-inoculated plants had shown higher abundance of antioxidant proteins, RuBisCo and ribosomal proteins that are important for enhancing stress tolerance and improving plant physiological traits. Collectively, salt stress might affect plant physiological traits by impairing photosynthetic machinery and accelerating apoptosis leading to a decline in biomass. However, inoculation of plants with bacteria can assist in enhancing photosynthetic activity, antioxidant activities and ethylene regulation related proteins for attenuating salt-induced apoptosis and sustaining growth and development.

ACC deaminase-producing Brevibacterium linens RS16 enhances heat-stress tolerance of rice (Oryza sativa L.).

The rapid rise in global temperature has adverse effects on rice productivity. The lack of eminent resources for heat stress alleviation is threatening the agricultural sector. Heat stress alleviation by endophytic plant growth-promoting bacteria (PGPB) can be a sustainable and eco-friendly approach. The present study was conducted to check the colonization of Brevibacterium linens RS16 producing ACC (1-aminocyclopropane-1-carboxylate) deaminase in the rice endosphere and to characterize its efficiency in enhancing stress tolerance. The ethylene emission pathway, reactive oxygen species (ROS) concentrations, proline accumulation, expression of glutathione S-transferase (GST), and small heat shock proteins (sHSPs) were monitored at two different levels of heat stress (40°C and 45°C). Bacterial inoculation decreased ethylene emission levels by 26.9% and 24.4% in rice plants exposed to 40°C and 45°C, respectively, compared with the non-inoculated plants. B. linens RS16 also enhanced the expression profiles of glutathione S-transferase. The collective effect of GST expression profiles and decrease in ethylene emission due to bacterial ACC deaminase activity subsequently resulted in a decrease in ROS concentrations. Additionally, HSP16 and HSP26 increased expression in heat-stressed plants inoculated with B. linens RS16 resulted in enhanced stress tolerance (i.e., lesser proline accumulation) than non-inoculated plants. Hence, this study demonstrates the bacteria-mediated tolerance against heat stress by regulating the ethylene emission pathway and upregulating antioxidant enzymes and heat shock proteins.

ACC deaminase and indole acetic acid producing endophytic bacterial co-inoculation improves physiological traits of red pepper (Capsicum annum L.) under salt stress.

Salinity induces myriad of physiological and biochemical perturbations in plants and its amelioration can be attained by the use of potential bacterial synthetic communities. The use of microbial consortia in contrast to single bacterial inoculation can additively enhance stress tolerance and productivity of agricultural crops. In this study, co-inoculation of Pseudomonas koreensis S2CB45 and Microbacterium hydrothermale IC37-36 isolated from arbuscular mycorrhizal fungi (AMF) spore and rice seed endosphere, respectively, were used to evaluate the physiological and biochemical effects on red pepper at two salt concentrations (75 mM and 150 mM). Plant growth promoting characteristics particularly 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, indole acetic acid (IAA) and cytokinin production were higher during co-culturing compared to the individual bacterial culture. The higher ACC deaminase activity had resulted in 20% and 22% decrease in stress ethylene emission compared to the non-inoculated plants at 75 mM and 150 mM salt stress, respectively. The decline in ethylene emission had eventually reduced ROS accumulation, and the co-inoculated plants had also harbored enhanced antioxidant enzyme activities and higher sugar accumulation compared to the other treatments suggesting enhanced tolerance to salinity. Collectively, these results put forward a novel consortium of bacterial strains that can be used for sustainable agricultural practices against salinity.

Conservation and transmission of seed bacterial endophytes across generations following crossbreeding and repeated inbreeding of rice at different geographic locations.

There are comparatively diverse bacterial communities inside seeds, which are vertically transmitted and conserved, becoming sources of endophytes in the next generation of host plants. We studied how rice seed endophyte composition changed over time following crossbreeding, repeated inbreeding, subsequent human selection and planting of different rice seeds in different ecogeographical locations. Using terminal-restriction fragment length polymorphism analysis to study bacterial communities, we observed that diversity between the original parents and their offspring may show significant differences in richness, evenness and diversity indices. Heat maps reveal substantial contributions of both or either parent in the shaping of the bacterial seed endophytes of the offspring. Most of the terminal restriction fragments (T-RFs) of the subsequent progeny could be traced to any or both of its parents while unique T-RFs of the offspring suggest external sources of colonization particularly when the seeds were cultivated in different locations. Many similar groups of endophytic bacteria persist in the seeds even after recultivation in different locations, indicating resilience to environmental changes and conservation of bacteria across generations. This study suggests that parent plants contributed to the shaping of seed bacterial endophytes of their offspring, although it is also possible that these soil grown rice plants recruit similar populations of endophytes from the soil generation after generation. This study also highlights some bacterial groups belonging to Herbaspirillum, Microbacterium, Curtobacterium, Stenotrophomonas, Xanthomonas and Enterobacter that may be part of a transmitted and conserved "core microbiota" that are ubiquitous and dominant members of the endophytic communities of the rice seeds.

The influence of host genotype and salt stress on the seed endophytic community of salt-sensitive and salt-tolerant rice cultivars.

BACKGROUND: Inherent characteristics and changes in the physiology of rice as it attains salt tolerance affect the colonizing bacterial endophytic communities of the rice seeds. These transmissible endophytes also serve as a source of the plant's microbial community and concurrently respond to the host and environmental conditions. This study explores the influence of the rice host as well as the impact of soil salinity on the community structure and diversity of seed bacterial endophytes of rice with varying tolerance to salt stress. Endophytic bacterial diversity was studied through culture-dependent technique and Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis. RESULTS: Results revealed considerably diverse communities of bacterial endophytes in the interior of rice seeds. The overall endophytic bacterial communities of the indica rice seeds based on 16S rRNA analysis of clones and isolates are dominated by phylum Proteobacteria followed by Actinobacteria and Firmicutes. Community profiles show common ribotypes found in all cultivars of the indica subspecies representing potential core microbiota belonging to Curtobacterium, Flavobacterium, Enterobacter, Xanthomonas, Herbaspirillum, Microbacterium and Stenotrophomonas. Clustering analysis shows that the host genotype mainly influences the seed endophytic community of the different rice cultivars. Under salt stress conditions, endophytic communities of the salt-sensitive and salt-tolerant rice cultivars shift their dominance to bacterial groups belonging to Flavobacterium, Pantoea, Enterobacter, Microbacterium, Kosakonia and Curtobacterium. CONCLUSION: The endophytic communities of rice indica seeds are shaped by the hosts' genotype, their physiological adaptation to salt stress and phylogenetic relatedness. Under salt stress conditions, a few groups of bacterial communities become prominent causing a shift in bacterial diversity and dominance.

Characterizing endophytic competence and plant growth promotion of bacterial endophytes inhabiting the seed endosphere of Rice.

BACKGROUND: Rice (Oryza sativa L. ssp. indica) seeds as plant microbiome present both an opportunity and a challenge to colonizing bacterial community living in close association with plants. Nevertheless, the roles and activities of bacterial endophytes remain largely unexplored and insights into plant-microbe interaction are compounded by its complexity. In this study, putative functions or physiological properties associated with bacterial endophytic nature were assessed. Also, endophytic roles in plant growth and germination that may allow them to be selectively chosen by plants were also studied. RESULTS: The cultivable seed endophytes were dominated by Proteobacteria particularly class Gammaproteobacteria. Highly identical type strains were isolated from the seed endosphere regardless of the rice host's physiological tolerance to salinity. Among the type strains, Flavobacterium sp., Microbacterium sp. and Xanthomonas sp. were isolated from the salt-sensitive and salt-tolerant cultivars. PCA-Biplot ordination also showed that specific type strains isolated from different rice cultivars have distinguishing similar characteristics. Flavobacterium sp. strains are phosphate solubilizers and indole-3-acetic acid producers with high tolerance to salinity and osmotic stress. Pseudomonas strains are characterized as high siderophore producers while Microbacterium sp. and Xanthomonas sp. strains have very high pectinase and cellulase activity. Among the physiological traits of the seed endophytes, bacterial pectinase and cellulase activity are positively correlated as well as salt and osmotic tolerance. Overall characterization shows that majority of the isolates could survive in 4-8% salt concentration as well as in 0.6 M and 1.2 M sucrose solution. The activities of catalase, pectinase and cellulase were also observed in almost all of the isolates indicating the importance of these characteristics for survival and colonization into the seed endosphere. Seed bacterial endophytes also showed promising plant growth promoting activities including hormone modulation, nitrogen fixation, siderophore production and phosphate solubilization. CONCLUSION: Though many of the isolates possess similar PGP and endophytic physiological traits, this study shows some prominent and distinguishing traits among bacterial groups indicating key determinants for their success as endophytes in the rice seed endosphere. Rice seeds are also inhabited by bacterial endophytes that promote growth during early seedling development.

Mycorrhizal Symbiotic Efficiency on C3 and C4 Plants under Salinity Stress - A Meta-Analysis.

A wide range of C3 and C4 plant species could acclimatize and grow under the impact of salinity stress. Symbiotic relationship between plant roots and arbuscular mycorrhizal fungi (AMF) are widespread and are well known to ameliorate the influence of salinity stress on agro-ecosystem. In the present study, we sought to understand the phenomenon of variability on AMF symbiotic relationship on saline stress amelioration in C3 and C4 plants. Thus, the objective was to compare varied mycorrhizal symbiotic relationship between C3 and C4 plants in saline conditions. To accomplish the above mentioned objective, we conducted a random effects models meta-analysis across 60 published studies. An effect size was calculated as the difference in mycorrhizal responses between the AMF inoculated plants and its corresponding control under saline conditions. Responses were compared between (i) identity of AMF species and AMF inoculation, (ii) identity of host plants (C3 vs. C4) and plant functional groups, (iii) soil texture and level of salinity and (iv) experimental condition (greenhouse vs. field). Results indicate that both C3 and C4 plants under saline condition responded positively to AMF inoculation, thereby overcoming the predicted effects of symbiotic efficiency. Although C3 and C4 plants showed positive effects under low (EC < 4 ds/m) and high (>8 ds/m) saline conditions, C3 plants showed significant effects for mycorrhizal inoculation over C4 plants. Among the plant types, C4 annual and perennial plants, C4 herbs and C4 dicot had a significant effect over other counterparts. Between single and mixed AMF inoculants, single inoculants Rhizophagus irregularis had a positive effect on C3 plants whereas Funneliformis mosseae had a positive effect on C4 plants than other species. In all of the observed studies, mycorrhizal inoculation showed positive effects on shoot, root and total biomass, and in nitrogen, phosphorous and potassium (K) uptake. However, it showed negative effects in sodium (Na) uptake in both C3 and C4 plants. This influence, owing to mycorrhizal inoculation, was significantly higher in K uptake in C4 plants. For our analysis, we concluded that AMF-inoculated C4 plants showed more competitive K(+) ions uptake than C3 plants. Therefore, maintenance of high cytosolic K(+)/Na(+) ratio is a key feature of plant salt tolerance. Studies on the detailed mechanism for the selective transport of K in C3 and C4 mycorrhizal plants under salt stress is lacking, and this needs to be explored.