Hai-Ping Cheng

Ascending Migration of Endophytic Rhizobia, from Roots to Leaves, inside Rice Plants and Assessment of Benefits to Rice Growth Physiology

ABSTRACT Rhizobia, the root-nodule endosymbionts of leguminous plants, also form natural endophytic associations with roots of important cereal plants. Despite its widespread occurrence, much remains unknown about colonization of cereals by rhizobia. We examined the infection, dissemination, and colonization of healthy rice plant tissues by four species of gfp-tagged rhizobia and their influence on the growth physiology of rice. The results indicated a dynamic infection process beginning with surface colonization of the rhizoplane (especially at lateral root emergence), followed by endophytic colonization within roots, and then ascending endophytic migration into the stem base, leaf sheath, and leaves where they developed high populations. In situ CMEIAS image analysis indicated local endophytic population densities reaching as high as 9 × 1010 rhizobia per cm3 of infected host tissues, whereas plating experiments indicated rapid, transient or persistent growth depending on the rhizobial strain and rice tissue examined. Rice plants inoculated with certain test strains of gfp-tagged rhizobia produced significantly higher root and shoot biomass; increased their photosynthetic rate, stomatal conductance, transpiration velocity, water utilization efficiency, and flag leaf area (considered to possess the highest photosynthetic activity); and accumulated higher levels of indoleacetic acid and gibberellin growth-regulating phytohormones. Considered collectively, the results indicate that this endophytic plant-bacterium association is far more inclusive, invasive, and dynamic than previously thought, including dissemination in both below-ground and above-ground tissues and enhancement of growth physiology by several rhizobial species, therefore heightening its interest and potential value as a biofertilizer strategy for sustainable agriculture to produce the worlds most important cereal crops.

Alfalfa Root Nodule Invasion Efficiency Is Dependent on Sinorhizobium meliloti Polysaccharides

The soil bacterium Sinorhizobium meliloti is capable of entering into a nitrogen-fixing symbiosis with Medicago sativa (alfalfa). Particular low-molecular-weight forms of certain polysaccharides produced by S. meliloti are crucial for establishing this symbiosis. Alfalfa nodule invasion by S. meliloti can be mediated by any one of three symbiotically important polysaccharides: succinoglycan, EPS II, or K antigen (also referred to as KPS). Using green fluorescent protein-labeled S. meliloti cells, we have shown that there are significant differences in the details and efficiencies of nodule invasion mediated by these polysaccharides. Succinoglycan is highly efficient in mediating both infection thread initiation and extension. However, EPS II is significantly less efficient than succinoglycan at mediating both invasion steps, and K antigen is significantly less efficient than succinoglycan at mediating infection thread extension. In the case of EPS II-mediated symbioses, the reduction in invasion efficiency results in stunted host plant growth relative to plants inoculated with succinoglycan or K-antigen-producing strains. Additionally, EPS II- and K-antigen-mediated infection threads are 8 to 10 times more likely to have aberrant morphologies than those mediated by succinoglycan. These data have important implications for understanding how S. meliloti polysaccharides are functioning in the plant-bacterium interaction, and models are discussed.

Sinorhizobium meliloti ExoR and ExoS Proteins Regulate both Succinoglycan and Flagellum Production

The production of the Sinorhizobium meliloti exopolysaccharide, succinoglycan, is required for the formation of infection threads inside root hairs, a critical step during the nodulation of alfalfa (Medicago sativa) by S. meliloti. Two bacterial mutations, exoR95::Tn5 and exoS96::Tn5, resulted in the overproduction of succinoglycan and a reduction in symbiosis. Systematic analyses of the symbiotic phenotypes of the two mutants demonstrated their reduced efficiency of root hair colonization. In addition, both the exoR95 and exoS96 mutations caused a marked reduction in the biosynthesis of flagella and consequent loss of ability of the cells to swarm and swim. Succinoglycan overproduction did not appear to be the cause of the suppression of flagellum biosynthesis. Further analysis indicated that both the exoR95 and exoS96 mutations affected the expression of the flagellum biosynthesis genes. These findings suggest that both the ExoR protein and the ExoS/ChvI two-component regulatory system are involved in the regulation of both succinoglycan and flagellum biosynthesis. These findings provide new avenues of understanding of the physiological changes S. meliloti cells go through during the early stages of symbiosis and of the signal transduction pathways that mediate such changes.

Two New Sinorhizobium meliloti LysR-Type Transcriptional Regulators Required for Nodulation

The establishment of an effective nitrogen-fixing symbiosis between Sinorhizobium meliloti and its legume host alfalfa (Medicago sativa) depends on the timely expression of nodulation genes that are controlled by LysR-type regulators. Ninety putative genes coding for LysR-type transcriptional regulators were identified in the recently sequenced S. meliloti genome. All 90 putative lysR genes were mutagenized using plasmid insertions as a first step toward determining their roles in symbiosis. Two new LysR-type symbiosis regulator genes, lsrA and lsrB, were identified in the screening. Both the lsrA and lsrB genes are expressed in free-living S. meliloti cells, but they are not required for cell growth. An lsrA1 mutant was defective in symbiosis and elicited only white nodules that exhibited no nitrogenase activity. Cells of the lsrA1 mutant were recovered from the white nodules, suggesting that the lsrA1 mutant was blocked early in nodulation. An lsrB1 mutant was deficient in symbiosis and elicited a mixture of pink and white nodules on alfalfa plants. These plants exhibited lower overall nitrogenase activity than plants inoculated with the wild-type strain, which is consistent with the fact that most of the alfalfa plants inoculated with the lsrB1 mutant were short and yellow. Cells of the lsrB1 mutant were recovered from both pink and white nodules, suggesting that lsrB1 mutants could be blocked at multiple points during nodulation. The identification of two new LysR-type symbiosis transcriptional regulators provides two new avenues for understanding the complex S. meliloti-alfalfa interactions which occur during symbiosis.

Identification of Sinorhizobium meliloti Early Symbiotic Genes by Use of a Positive Functional Screen

ABSTRACT The soil bacterium Sinorhizobium meliloti establishes nitrogen-fixing symbiosis with its leguminous host plant, alfalfa, following a series of continuous signal exchanges. The complexity of the changes of alfalfa root structures during symbiosis and the amount of S. meliloti genes with unknown functions raised the possibility that more S. meliloti genes may be required for early stages of the symbiosis. A positive functional screen of the entire S. meliloti genome for symbiotic genes was carried out using a modified in vivo expression technology. A group of genes and putative genes were found to be expressed in early stages of the symbiosis, and 23 of them were alfalfa root exudate inducible. These 23 genes were further separated into two groups based on their responses to apigenin, a known nodulation (nod) gene inducer. The group of six genes not inducible by apigenin included the lsrA gene, which is essential for the symbiosis, and the dgkA gene, which is involved in the synthesis of cyclic β-1,2-glucan required for the S. meliloti-alfalfa symbiosis. In the group of 17 apigenin-inducible genes, most have not been previously characterized in S. meliloti, and none of them belongs to the nod gene family. The identification of this large group of alfalfa root exudate-inducible S. meliloti genes suggests that the interactions in the early stages of the S. meliloti and alfalfa symbiosis could be complex and that further characterization of these genes will lead to a better understanding of the symbiosis.

Sinorhizobium meliloti ExoR Is the Target of Periplasmic Proteolysis

Sinorhizobium meliloti ExoR regulates the production of succinoglycan and flagella through the ExoS/ChvI two-component regulatory system. ExoR has been proposed to inhibit the ExoS sensor through direct interaction in the periplasm. To understand how ExoR suppression of ExoS is relieved, which is required for the expression of ExoS/ChvI-regulated symbiosis genes, we characterized wild-type ExoR and ExoR95 mutant proteins. In addition to the previously identified precursor and mature forms of ExoR (designated ExoR(p) and ExoR(m), respectively), we detected a 20-kDa form of ExoR (designated ExoR(c20)) derived from the wild-type ExoR protein, but not from the ExoR95 mutant protein. ExoR(c20) was isolated directly from S. meliloti periplasm to identify its N-terminal amino acids and the site of the proteolysis, which is highly conserved among ExoR homologs. ExoR(c20) retains the C terminus of the wild-type ExoR. When expressed directly, ExoR(c20) did not complement the exoR95 mutation, suggesting that ExoR(c20) does not function directly in the ExoR-ExoS/ChvI regulatory pathway and that ExoR(m) is the functional form of ExoR. A single-amino-acid change (ExoRL81A) at the site of ExoR periplasmic proteolysis resulted in the reduction of the amount of ExoR(m) and the loss of the regulatory function of the ExoR protein. These findings suggest that ExoR(m) is a target of periplasmic proteolysis and that the amount of ExoR(m) could be reduced through effective proteolysis to relieve its suppression of ExoS.

Autoregulation of Sinorhizobium meliloti exoR gene expression

The successful nitrogen-fixing symbiosis between the Gram-negative soil bacterium Sinorhizobium meliloti and its leguminous plant host alfalfa (Medicago sativa) requires the bacterial exopolysaccharide succinoglycan. Succinoglycan and flagellum production, along with the ability to metabolize more than 20 different carbon sources and control the expression of a large number of S. meliloti genes, is regulated by the ExoR–ExoS/ChvI signalling pathway. The ExoR protein interacts with and suppresses the sensing activities of ExoS, the membrane-bound sensor of the ExoS/ChvI two-component regulatory system. Here we show that exoR expression is clearly upregulated in the absence of any functional ExoR protein. This upregulation was suppressed by the presence of the wild-type ExoR protein but not by a mutated ExoR protein lacking signal peptide. The levels of exoR expression could be directly modified in real time by changing the levels of total ExoR protein. The expression of exoR was also upregulated by the constitutively active sensor mutation exoS96, and blocked by two single mutations, exoS* and exoSsupA, in the ExoS sensing domain. Presence of the wild-type ExoS protein further elevated the levels of exoR expression in the absence of functional ExoR protein, and reversed the effects of exoS96, exoS* and exoSsupA mutations. Altogether, these data suggest that ExoR protein autoregulates exoR expression through the ExoS/ChvI system, allowing S. meliloti cells to maintain the levels of exoR expression based on the amount of total ExoR protein.

Phylogenetic Co-Occurrence of ExoR, ExoS, and ChvI, Components of the RSI Bacterial Invasion Switch, Suggests a Key Adaptive Mechanism Regulating the Transition between Free-Living and Host-Invading Phases in Rhizobiales

Both bacterial symbionts and pathogens rely on their host-sensing mechanisms to activate the biosynthetic pathways necessary for their invasion into host cells. The Gram-negative bacterium Sinorhizobium meliloti relies on its RSI (ExoR-ExoS-ChvI) Invasion Switch to turn on the production of succinoglycan, an exopolysaccharide required for its host invasion. Recent whole-genome sequencing efforts have uncovered putative components of RSI-like invasion switches in many other symbiotic and pathogenic bacteria. To explore the possibility of the existence of a common invasion switch, we have conducted a phylogenomic survey of orthologous ExoR, ExoS, and ChvI tripartite sets in more than ninety proteobacterial genomes. Our analyses suggest that functional orthologs of the RSI invasion switch co-exist in Rhizobiales, an order characterized by numerous invasive species, but not in the order’s close relatives. Phylogenomic analyses and reconstruction of orthologous sets of the three proteins in Alphaproteobacteria confirm Rhizobiales-specific gene synteny and congruent RSI evolutionary histories. Evolutionary analyses further revealed site-specific substitutions correlated specifically to either animal-bacteria or plant-bacteria associations. Lineage restricted conservation of any one specialized gene is in itself an indication of species adaptation. However, the orthologous phylogenetic co-occurrence of all interacting partners within this single signaling pathway strongly suggests that the development of the RSI switch was a key adaptive mechanism. The RSI invasion switch, originally found in S. meliloti, is a characteristic of the Rhizobiales, and potentially a conserved crucial activation step that may be targeted to control host invasion by pathogenic bacterial species.

Ascending Migration of Endophytic Rhizobia from Roots to Leaves Inside Rice Plants

Rhizobia, the root-nodule endosymbionts of leguminous plants, also form natural endophytic associations with roots of important cereal plants. Despite its widespread occurrence, much remains unknown about colonization of cereals by rhizobia. We examined the infection, dissemination, and colonization of healthy rice plant tissues by four species of gfp-tagged rhizobia and their influence on the growth physiology of rice. The results indicated a dynamic infection process beginning with surface colonization of the rhizoplane (especially at lateral root emergence), followed by endophytic colonization within roots, and then ascending endophytic migration into the stem base, leaf sheath, and leaves where they developed high populations. In situ CMEIAS image analysis indicated local endophytic population densities reaching as high as 9 x 10(10) rhizobia per cm3 of infected host tissues, whereas plating experiments indicated rapid, transient or persistent growth depending on the rhizobial strain and rice tissue examined. Rice plants inoculated with certain test strains of gfp-tagged rhizobia produced significantly higher root and shoot biomass; increased their photosynthetic rate, stomatal conductance, transpiration velocity, water utilization efficiency, and flag leaf area (considered to possess the highest photosynthetic activity); and accumulated higher levels of indoleacetic acid and gibberellin growth-regulating phytohormones. Considered collectively, the results indicate that this endophytic plant-bacterium association is far more inclusive, invasive, and dynamic than previously thought, including dissemination in both below-ground and above-ground tissues and enhancement of growth physiology by several rhizobial species, therefore heightening its interest and potential value as a biofertilizer strategy for sustainable agriculture to produce the worlds most important cereal crops.

Isolation of Two New Sinorhizobium meliloti Transcriptional Regulators Required for Nodulation

In the absence of fixed nitrogen, Sinorhizobium meliloti establishes an effective nitrogen fixing symbiosis with its legume partner, alfalfa (Medicago sativa) through a series of intricate signal exchanges and reciprocal structural changes. The signal exchanges between the two organisms starts with the alfalfa flavonoids and the S. meliloti nodulation factors. This exchange of signals results in the formation of tightly curled alfalfa root hairs. S. meliloti cells elicit the formation of infection threads inside these curled root hairs. The infection threads elongate toward the base of root hairs, penetrating layers of alfalfa root cells, reaching developing nodule primordium, and releasing live bacterial cells into the plant cells inside the nodule primordium. The successful differentiation of the S. meliloti cells may depend on another set of signal exchanges between S. meliloti and alfalfa involving proteins like BacA. The complexity of the S. meliloti-alfalfa interaction raises the possibility that many more signal exchanges between the two organisms remain unknown. Some of the key regulatory genes involved in the signal exchanges between S. meliloti and alfalfa belong to the family of LysR transcriptional regulators. The production of nodulation factor that is part of the signal exchanges that initiate symbiosis, is positively controlled by three different nodD genes: nodD1, nodD2 and nodD3 and the syrM gene. The nod factor production is also negatively regulated by the nolR gene (9). The nolR gene encodes a repressor that negatively regulates the expression of the nodD1 and nodABC operons by binding to the divergent promoter between them. The genomic sequence has been determined for S. meliloti Rm1021 and 90 putative lysR family transcriptional regulators have been identified. Each of 90 previously unknown putative S. meliloti lysR genes was interrupted by insertions of suicide plasmids into the middle of the open reading frames.