Rhizobacterium-derived diacetyl modulates plant immunity in a phosphate-dependent manner.

Plants establish mutualistic associations with beneficial microbes while deploying the immune system to defend against pathogenic ones. Little is known about the interplay between mutualism and immunity and the mediator molecules enabling such crosstalk. Here, we show that plants respond differentially to a volatile bacterial compound through integral modulation of the immune system and the phosphate-starvation response (PSR) system, resulting in either mutualism or immunity. We found that exposure of Arabidopsis thaliana to a known plant growth-promoting rhizobacterium can unexpectedly have either beneficial or deleterious effects to plants. The beneficial-to-deleterious transition is dependent on availability of phosphate to the plants and is mediated by diacetyl, a bacterial volatile compound. Under phosphate-sufficient conditions, diacetyl partially suppresses plant production of reactive oxygen species (ROS) and enhances symbiont colonization without compromising disease resistance. Under phosphate-deficient conditions, diacetyl enhances phytohormone-mediated immunity and consequently causes plant hyper-sensitivity to phosphate deficiency. Therefore, diacetyl affects the type of relation between plant hosts and certain rhizobacteria in a way that depends on the plant's phosphate-starvation response system and phytohormone-mediated immunity.

Flavonoid-attracted Aeromonas sp. from the Arabidopsis root microbiome enhances plant dehydration resistance.

Flavonoids are stress-inducible metabolites important for plant-microbe interactions. In contrast to their well-known function in initiating rhizobia nodulation in legumes, little is known about whether and how flavonoids may contribute to plant stress resistance through affecting non-nodulating bacteria. Here we show that flavonoids broadly contribute to the diversity of the Arabidopsis root microbiome and preferentially attract Aeromonadaceae, which included a cultivable Aeromonas sp. H1 that displayed flavonoid-induced chemotaxis with transcriptional enhancement of flagellum biogenesis and suppression of fumarate reduction for smooth swims. Strain H1 showed multiple plant-beneficial traits and enhanced plant dehydration resistance, which required flavonoids but not through a sudden "cry-for-help" upon stress. Strain H1 boosted dehydration-induced H2O2 accumulation in guard cells and stomatal closure, concomitant with synergistic induction of jasmonic acid-related regulators of plant dehydration resistance. These findings revealed a key role of flavonoids, and the underlying mechanism, in mediating plant-microbiome interactions including the bacteria-enhanced plant dehydration resistance.

Plant latent defense response to microbial non-pathogenic factors antagonizes compatibility.

Unlike microbe-associated molecular patterns (MAMPs) that are readily targeted by host immunity, microbial non-pathogenic factors (NPFs) appear negligible as they do not elicit defense. Little is known about whether and how NPFs may be monitored by hosts to control compatibility. Herein, a forward genetic screening isolated an Arabidopsis mutant with a loss of plant-rhizobacteria mutualism, leading to the disclosure of a plant latent defense response (LDR) to NPFs. The activation of LDR in the mutant, named rol1 for regulator of LDR 1, is triggered by several non-pathogenic volatile organic compounds and antagonizes plant compatibility with the beneficial bacterium Bacillus amyloliquefaciens GB03. The activation of LDR in rol1 is mediated through the prokaryotic pathway of chloroplastic lipid biosynthesis. The rol1 root microbiome showed a reduced proportion of the Bacillaceae family. We propose that, parallel to the forefront immunity to MAMPs, LDR to certain NPFs provides a hidden layer of defense for controlling compatibility with commensal or beneficial microbes.

Plant Transcriptome Reprograming and Bacterial Extracellular Metabolites Underlying Tomato Drought Resistance Triggered by a Beneficial Soil Bacteria.

Water deficit is one of the major constraints to crop production and food security worldwide. Some plant growth-promoting rhizobacteria (PGPR) strains are capable of increasing plant drought resistance. Knowledge about the mechanisms underlying bacteria-induced plant drought resistance is important for PGPR applications in agriculture. In this study, we show the drought stress-mitigating effects on tomato plants by the Bacillus megaterium strain TG1-E1, followed by the profiling of plant transcriptomic responses to TG1-E1 and the profiling of bacterial extracellular metabolites. Comparison between the transcriptomes of drought-stressed plants with and without TG1-E1 inoculation revealed bacteria-induced transcriptome reprograming, with highlights on differentially expressed genes belonging to the functional categories including transcription factors, signal transduction, and cell wall biogenesis and organization. Mass spectrometry-based analysis identified over 40 bacterial extracellular metabolites, including several important regulators or osmoprotectant precursors for increasing plant drought resistance. These results demonstrate the importance of plant transcriptional regulation and bacterial metabolites in PGPR-induced plant drought resistance.

Dicer-like proteins influence Arabidopsis root microbiota independent of RNA-directed DNA methylation.

BACKGROUND: Plants are naturally associated with root microbiota, which are microbial communities influential to host fitness. Thus, it is important to understand how plants control root microbiota. Epigenetic factors regulate the readouts of genetic information and consequently many essential biological processes. However, it has been elusive whether RNA-directed DNA methylation (RdDM) affects root microbiota assembly. RESULTS: By applying 16S rRNA gene sequencing, we investigated root microbiota of Arabidopsis mutants defective in the canonical RdDM pathway, including dcl234 that harbors triple mutation in the Dicer-like proteins DCL3, DCL2, and DCL4, which produce small RNAs for RdDM. Alpha diversity analysis showed reductions in microbe richness from the soil to roots, reflecting the selectivity of plants on root-associated bacteria. The dcl234 triple mutation significantly decreases the levels of Aeromonadaceae and Pseudomonadaceae, while it increases the abundance of many other bacteria families in the root microbiota. However, mutants of the other examined key players in the canonical RdDM pathway showed similar microbiota as Col-0, indicating that the DCL proteins affect root microbiota in an RdDM-independent manner. Subsequently gene analysis by shotgun sequencing of root microbiome indicated a selective pressure on microbial resistance to plant defense in the dcl234 mutant. Consistent with the altered plant-microbe interactions, dcl234 displayed altered characters, including the mRNA and sRNA transcriptomes that jointly highlighted altered cell wall organization and up-regulated defense, the decreased cellulose and callose deposition in root xylem, and the restructured profile of root exudates that supported the alterations in gene expression and cell wall modifications. CONCLUSION: Our findings demonstrate an important role of the DCL proteins in influencing root microbiota through integrated regulation of plant defense, cell wall compositions, and root exudates. Our results also demonstrate that the canonical RdDM is dispensable for Arabidopsis root microbiota. These findings not only establish a connection between root microbiota and plant epigenetic factors but also highlight the complexity of plant regulation of root microbiota. Video abstract.

Bacteria-derived diacetyl enhances Arabidopsis phosphate starvation responses partially through the DELLA-dependent gibberellin signaling pathway.

Plant growth-promoting rhizobacteria (PGPR) are naturally occurring soil microorganisms that colonize roots and stimulate plant growth. Some PGPR strains can directly regulate plant growth in the absence of physical contact with the plant, via volatile organic compounds (VOCs) emissions. Recently, we have described that Arabidopsis thaliana respond differentially to diacetyl, a VOC from Bacillus amyloliquefaciens strain GB03 (GB03), through integral modulation of the immune system and the phosphate-starvation response (PSR) system, resulting in either mutualism or immunity. Under phosphate deficient conditions, diacetyl enhances salicylic acid- and jasmonic acid-mediated immunity and consequently causes plant hyper-sensitivity to phosphate deficiency. Here, we show that application of exogenous gibberellin (GA) partially alleviates the deleterious effect caused by either B. amyloliquefaciens GB03 VOCs or diacetyl in Arabidopsis under phosphate deficient conditions, while DELLA quadruple mutant exposed to GB03 VOCs exhibits a partial reduction on the stress symptoms. Moreover, diacetyl appears to enhance DELLA protein accumulation and increase the expression of several GA deactivation-related genes. These findings suggest that the DELLA-mediated GA signaling pathway is involved in the bi-faceted role of GB03 VOCs in regulating plant growth.

DNA demethylases are required for myo-inositol-mediated mutualism between plants and beneficial rhizobacteria.

Root-associated soil bacteria can strongly influence plant fitness. DNA methylation is an epigenetic mark important to many fundamental biological processes; however, its roles in plant interactions with beneficial microbes remain elusive. Here, we report that active DNA demethylation in Arabidopsis controls root secretion of myo-inositol and consequently plant growth promotion triggered by Bacillus megaterium strain YC4. Root-secreted myo-inositol is critical for YC4 colonization and preferentially attracts B. megaterium among the examined bacteria species. Active DNA demethylation antagonizes RNA-directed DNA methylation in controlling myo-inositol homeostasis. Importantly, we demonstrate that active DNA demethylation controls myo-inositol-mediated mutualism between YC4 and Solanum lycopersicum, thus suggesting a conserved nature of this epigenetic regulatory mechanism.

The involvement of McpB chemoreceptor from Pseudomonas aeruginosa PAO1 in virulence.

Pseudomonas aeruginosa is an opportunistic human pathogen causing infections in a variety of plant and animal hosts. The gene mcpB, part of the chemosensory gene cluster II, encodes a soluble chemoreceptor whose function remains unknown. Previous studies show that the cheB2 gene, also located in the chemosensory cluster II, is involved in a specific response during infection and it is required for full pathogenicity of P. aeruginosa. To determine whether the McpB (or Aer2) chemoreceptor is involved in virulence processes, we generated a mcpB mutant and tested its phenotype using a virulence-measuring system. This system was developed by our group and is based on different bioassays using organisms living at different soil trophic levels, including microbial, nematode, arthropod, annelid, and plant model systems. The deletion of mcpB resulted in an attenuation of bacterial virulence in different infection models, and wild-type virulence was restored following genetic complementation of the mutant strain. Our study indicates that the McpB chemoreceptor is linked to virulence processes and may constitute the basis for the development of alternative strategies against this pathogen.

Re-addressing the biosafety issues of plant growth promoting rhizobacteria.

To promote agronomic sustainability, extensive research is being carried out globally, investigating biofertilizer development. Recently, it has been realized that some microorganisms used as biofertilizers behave as opportunistic pathogens and belong to the biosafety level 2 (BSL-2) classification. This poses serious risk to the environmental and human health. Evidence presented in various scientific forums is increasingly favoring the merits of using BSL-2 microorganisms as biofertilizers. In this review, we emphasize that partial characterization based on traditional microbiological approaches and small subunit rRNA gene sequences/conserved regions are insufficient for the characterization of biofertilizer strains. It is advised herein, that research and industrial laboratories developing biofertilizers for commercialization or environmental release must characterize microorganisms of interest using a multilateral polyphasic approach of microbial systematics. This will determine their risk group and biosafety characteristics before proceeding with formulation development and environmental application. It has also been suggested that microorganisms belonging to risk-group-1 and BSL-1 category should be used for formulation development and for field scale applications. While, BSL-2 microorganisms should be restricted for research using containment practices compliant with strict regulations.

Protection of Pepper Plants from Drought by Microbacterium sp. 3J1 by Modulation of the Plant's Glutamine and α-ketoglutarate Content: A Comparative Metabolomics Approach.

Drought tolerance of plants such as tomato or pepper can be improved by their inoculation with rhizobacteria such as Microbacterium sp. 3J1. This interaction depends on the production of trehalose by the microorganisms that in turn modulate the phyto-hormone profile of the plant. In this work we describe the characterization of metabolic changes during the interaction of pepper plants with Microbacterium sp. 3J1 and of the microorganism alone over a period of drought. Our main findings include the observation that the plant responds to the presence of the microorganism by changing the C and N metabolism based on its glutamine and α-ketoglutarate content, these changes contribute to major changes in the concentration of molecules involved in the balance of the osmotic pressure. These include sugars and amino-acids; the concentration of antioxidant molecules, of metabolites involved in the production of phytohormones like ethylene, and of substrates used for lignin production such as ferulic and sinapic acids. Most of the altered metabolites of the plant when inoculated with Microbacterium sp. 3J1 in response to drought coincided with the profile of altered metabolites in the microorganism alone when subjected to drought, pointing to a response by which the plant relies on the microbe for the production of such metabolites. To our knowledge this is the first comparative study of the microbe colonized-plant and microbe alone metabolomes under drought stress.

Plant Drought Tolerance Enhancement by Trehalose Production of Desiccation-Tolerant Microorganisms.

A collection of desiccation-tolerant xeroprotectant-producing microorganisms was screened for their ability to protect plants against drought, and their role as plant growth-promoting rhizobacteria was investigated in two different crops (tomato and pepper). The most commonly described biochemical mechanisms for plant protection against drought by microorganisms including the production of phytohormones, antioxidants and xeroprotectants were analyzed. In particular, the degree of plant protection against drought provided by these microorganisms was characterized. After studying the findings and comparing them with results of the closest taxonomic relatives at the species and strain levels, we propose that trehalose produced by these microorganisms is correlated with their ability to protect plants against drought. This proposal is based on the increased protection of plants against drought by the desiccation-sensitive microorganism Pseudomonas putida KT2440, which expresses the otsAB genes for trehalose biosynthesis in trans.

Biosafety Test for Plant Growth-Promoting Bacteria: Proposed Environmental and Human Safety Index (EHSI) Protocol.

Plant growth-promoting bacteria (PGPB) colonize plants and enhance their growth by different mechanisms. Some of these microorganisms may represent a potential threat to human, animal or plant health; however, their use might be approved in parts of Europe if they have been recommended as plant growth enhancers. The current regulatory framework has resulted in a fragmented, contradictory system, and there is an urgent need to establish harmonized protocols for the predictability, efficiency, consistency and especially the safety of PGPB for human and animal health and for the environment. In response to current efforts to update biosafety policies and provide alternative methods to replace the use of vertebrate animals, we propose a panel of tests and an evaluation system to reliably determine the biosafety of bacterial strains used as PGPB. Based on the results of different tests, we propose a scoring system to evaluate the safety of candidates for PGPB within the limitations of the assays used.