Tracing My Roots: How I Became a Plant Biologist.

My trajectory to becoming a plant biologist was shaped by a complex mix of scientific, political, sociological, and personal factors. I was trained as a microbiologist and molecular biologist in the late 1960s and early 1970s, a time of political upheaval surrounding the Vietnam War. My political activism taught me to be wary of the potential misuses of scientific knowledge and to promote the positive applications of science for the benefit of society. I chose agricultural science for my postdoctoral work. Because I was not trained as a plant biologist, I devised a postdoctoral project that took advantage of my microbiological training, and I explored using genetic technologies to transfer the ability to fix nitrogen from prokaryotic nitrogen-fixing species to the model plant Arabidopsis thaliana with the ultimate goal of engineering crop plants. The invention of recombinant DNA technology greatly facilitated the cloning and manipulation of bacterial nitrogen-fixation ( nif) genes, but it also forced me to consider how much genetic engineering of organisms, including human beings, is acceptable. My laboratory has additionally studied host-pathogen interactions using Arabidopsis and the nematode Caenorhabditis elegans as model hosts.

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.

[Influence of salt stress on the genetically polymorphic system of Sinorhizobium meliloti-Medicago truncatula].

The impacts of salt stress (75 mM NaC1) on the ecological efficiency of the genetically polymorphic Sinorhizobium meliloti-Medicago truncatula system were studied. Its impact on a symbiotic system results in an increase of the partners' variability for symbiotic traits and of the symbiosis integrity as indicated by: a) the specificity of the partners' interactions--the nonadditive inputs of their genotypes into the variation of symbiotic parameters; and b) the correlative links between these parameters. The structure of the nodDI locus and the content correlates to the efficiency of the symbiosis between S. meliloti and M. truncatula genotypes under stress conditions more sufficiently than in the absence of stress. Correlations between the symbiotic efficiency of rhizobia strains and their growth rate outside symbiosis are expressed under stress conditions, not in the absence of stress. Under salt stress symbiotic effectiveness was decreased for M. truncatula line F83005.5, which was salt sensitive for mineral nutrition. The inhibition of symbiotic activity for this line is linked with decreased nodule formation, whereas for Jemalong 6 and DZA315.16 lines it is associated with repressed N2-fixation. It was demonstrated for the first time that salt stress impairs the M. truncatula habitus (the mass : height ratio increased 2- to 6-fold), which in the salt-resistant cultivar Jemalong 6 is normalized as the result of rhizobia inoculation.

Plant-activated bacterial receptor adenylate cyclases modulate epidermal infection in the Sinorhizobium meliloti-Medicago symbiosis.

Legumes and soil bacteria called rhizobia have coevolved a facultative nitrogen-fixing symbiosis. Establishment of the symbiosis requires bacterial entry via root hair infection threads and, in parallel, organogenesis of nodules that subsequently are invaded by bacteria. Tight control of nodulation and infection is required to maintain the mutualistic character of the interaction. Available evidence supports a passive bacterial role in nodulation and infection after the microsymbiont has triggered the symbiotic plant developmental program. Here we identify in Sinorhizobium meliloti, the Medicago symbiont, a cAMP-signaling regulatory cascade consisting of three receptor-like adenylate cyclases, a Crp-like regulator, and a target gene of unknown function. The cascade is activated specifically by a plant signal during nodule organogenesis. Cascade inactivation results in a hyperinfection phenotype consisting of abortive epidermal infection events uncoupled from nodulation. These findings show that, in response to a plant signal, rhizobia play an active role in the control of infection. We suggest that rhizobia may modulate the plant's susceptibility to infection. This regulatory loop likely aims at optimizing legume infection.

Symbiotic rhizobia bacteria trigger a change in localization and dynamics of the Medicago truncatula receptor kinase LYK3.

To form nitrogen-fixing symbioses, legume plants recognize a bacterial signal, Nod Factor (NF). The legume Medicago truncatula has two predicted NF receptors that direct separate downstream responses to its symbiont Sinorhizobium meliloti. NOD FACTOR PERCEPTION encodes a putative low-stringency receptor that is responsible for calcium spiking and transcriptional responses. LYSIN MOTIF RECEPTOR-LIKE KINASE3 (LYK3) encodes a putative high-stringency receptor that mediates bacterial infection. We localized green fluorescent protein (GFP)-tagged LYK3 in M. truncatula and found that it has a punctate distribution at the cell periphery consistent with a plasma membrane or membrane-tethered vesicle localization. In buffer-treated control roots, LYK3:GFP puncta are dynamic. After inoculation with compatible S. meliloti, LYK3:GFP puncta are relatively stable. We show that increased LYK3:GFP stability depends on bacterial NF and NF structure but that NF is not sufficient for the change in LYK3:GFP dynamics. In uninoculated root hairs, LYK3:GFP has little codistribution with mCherry-tagged FLOTILLIN4 (FLOT4), another punctate plasma membrane-associated protein required for infection. In inoculated root hairs, we observed an increase in FLOT4:mCherry and LYK3:GFP colocalization; both proteins localize to positionally stable puncta. We also demonstrate that the localization of tagged FLOT4 is altered in plants carrying a mutation that inactivates the kinase domain of LYK3. Our work indicates that LYK3 protein localization and dynamics are altered in response to symbiotic bacteria.

Plant flotillins are required for infection by nitrogen-fixing bacteria.

To establish compatible rhizobial-legume symbioses, plant roots support bacterial infection via host-derived infection threads (ITs). Here, we report the requirement of plant flotillin-like genes (FLOTs) in Sinorhizobium meliloti infection of its host legume Medicago truncatula. Flotillins in other organisms have roles in viral pathogenesis, endocytosis, and membrane shaping. We identified seven FLOT genes in the M. truncatula genome and show that two, FLOT2 and FLOT4, are strongly up-regulated during early symbiotic events. This up-regulation depends on bacterial Nod Factor and the plant's ability to perceive Nod Factor. Microscopy data suggest that M. truncatula FLOT2 and FLOT4 localize to membrane microdomains. Upon rhizobial inoculation, FLOT4 uniquely becomes localized to the tips of elongating root hairs. Silencing FLOT2 and FLOT4 gene expression reveals a nonredundant requirement for both genes in IT initiation and nodule formation. FLOT4 is uniquely required for IT elongation, and FLOT4 localizes to IT membranes. This work reveals a critical role for plant flotillins in symbiotic bacterial infection.

Strain competition and agar affect the interaction of rhizobia with rice.

Competition assays with Sinorhizobium meliloti 1021 and its GFP-labelled pSymA cured and deleted derivatives, SmA818 and SmA146, demonstrated that Sm1021 could still inhibit rice seedling growth even when outnumbered by a large excess of the noninhibitory cured or deleted strain. The wild-type strain Sm1021 also inhibited the growth of its noninhibitory pSymA-cured strain SmA818(gfp) and its pSymA-deleted strain SmA146(gfp) in a manner suggesting that Sm1021 produced a bacteriocin-like substance. The production of, and resistance to, this substance seemed to be pSymA-associated, but it was not the cause of killing in competition experiments on rice, suggesting that the killing of SmA818(gfp) and SmA146(gfp) was medium dependent. The addition of agar in liquid F10 medium at concentrations < or = 0.4% (m/v) abolished the rice growth inhibition of strain Sm1021 and Sm1021(gfp). The increased medium viscosity at higher agar concentrations decreased the diffusion of gases and small molecules through the media. Thus, the low agar concentrations may mimic waterlogged soil conditions leading to the production of inhibitory compounds by the bacterial strains under microaerobic conditions.

The type IV secretion system of Sinorhizobium meliloti strain 1021 is required for conjugation but not for intracellular symbiosis.

The type IV secretion system (T4SS) of the plant intracellular symbiont Sinorhizobium meliloti 1021 is required for conjugal transfer of DNA. However, it is not required for host invasion and persistence, unlike the T4SSs of closely related mammalian intracellular pathogens. A comparison of the requirement for a bacterial T4SS in plant versus animal host invasion suggests an important difference in the intracellular niches occupied by these bacteria.

QSARS for toxicity to the bacterium Sinorhizobium meliloti.

In the present study, structure-activity relationship (QSAR) models for the prediction of the toxicity to the bacterium Sinorhizobium meliloti have been developed, based on a data set of 140 compounds. The data set is highly heterogeneous both in terms of chemistry and mechanisms of toxic action. For deriving QSARs, chemicals were divided into groups according to mechanism of action and chemical structure. The QSARs derived are considered to be of moderate statistical quality. A baseline effect (relationship between the toxicity and logP), which can be related to non-polar narcosis, was observed. To explain toxicity greater than the baseline toxicity, other structural descriptors were used. The development of models for non-polar and polar narcosis had some success. It appeared that the toxicity of compounds acting by more specific mechanisms of toxic action is difficult to predict. A global QSAR was also developed, which had square of the correlation coefficient r2 = 0.53. A QSAR with reasonable statistical parameters was developed for the aliphatic compounds in the data set (r2 = 0.83). QSARs could not be obtained for the aromatic compounds as a group.

Altered susceptibility to infection by Sinorhizobium meliloti and Nectria haematococca in alfalfa roots with altered cell cycle.

Most infections of plant roots are initiated in the region of elongation; the mechanism for this tissue-specific localization pattern is unknown. In alfalfa expressing PsUGT1 antisense mRNA under the control of the cauliflower mosaic virus (CaMV) 35S promoter, the cell cycle in roots is completed in 48 h instead of 24 h, and border cell number is decreased by more than 99%. These plants were found to exhibit increased root-tip infection by a fungal pathogen and reduced nodule formation by a bacterial symbiont. Thus, the frequency of infection in the region of elongation by Nectria haematocca was unaffected, but infection of the root tip was increased by more than 90%; early stages of Sinorhizobium meliloti infection and nodule morphology were normal, but the frequency of nodulation was fourfold lower than in wild-type roots.

Molecular sensing of bacteria in plants. The highly conserved RNA-binding motif RNP-1 of bacterial cold shock proteins is recognized as an elicitor signal in tobacco.

To detect microbial infection multicellular organisms have evolved sensing systems for pathogen-associated molecular patterns (PAMPs). Here, we identify bacterial cold shock protein (CSP) as a new such PAMP that acts as a highly active elicitor of defense responses in tobacco. Tobacco cells perceive a conserved domain of CSP and synthetic peptides representing 15 amino acids of this domain-induced responses at subnanomolar concentrations. Central to the elicitor-active domain is the RNP-1 motif KGFGFITP, a motif conserved also in many RNA- and DNA-binding proteins of eukaryotes. Csp15-Nsyl, a peptide representing the domain with highest homology to csp15 in a protein of Nicotiana sylvestris exhibited only weak activity in tobacco cells. Crystallographic and genetic data from the literature show that the RNP-1 domain of bacterial CSPs resides on a protruding loop and exposes a series of aromatic and basic side chains to the surface that are essential for the nucleotide-binding activity of CSPs. Similarly, these side chains were also essential for elicitor activity and replacement of single residues in csp15 with Ala strongly reduced or abolished activity. Most strikingly, csp15-Ala10, a peptide with the RNP-1 motif modified to KGAGFITP, lacked elicitor activity but acted as a competitive antagonist for CSP-related elicitors. Bacteria commonly have a small family of CSP-like proteins including both cold-inducible and noninducible members, and Csp-related elicitor activity was detected in extracts from all bacteria tested. Thus, the CSP domain containing the RNP-1 motif provides a structure characteristic for bacteria in general, and tobacco plants have evolved a highly sensitive chemoperception system to detect this bacterial PAMP.

The Sinorhizobium meliloti stringent response affects multiple aspects of symbiosis.

Sinorhizobium meliloti and host legumes enter into a nitrogen-fixing, symbiotic relationship triggered by an exchange of signals between bacteria and plant. S. meliloti produces Nod factor, which elicits the formation of nodules on plant roots, and succinoglycan, an exopolysaccharide that allows for bacterial invasion and colonization of the host. The biosynthesis of these molecules is well defined, but the specific regulation of these compounds is not completely understood. Bacteria control complex regulatory networks by the production of ppGpp, the effector molecule of the stringent response, which induces physiological change in response to adverse growth conditions and can also control bacterial development and virulence. Through detailed analysis of an S. meliloti mutant incapable of producing ppGpp, we show that the stringent response is required for nodule formation and regulates the production of succinoglycan. Although it remains unknown whether these phenotypes are connected, we have isolated suppressor strains that restore both defects and potentially identify key downstream regulatory genes. These results indicate that the S. meliloti stringent response has roles in both succinoglycan production and nodule formation and, more importantly, that control of bacterial physiology in response to the plant and surrounding environment is critical to the establishment of a successful symbiosis.

Cell cycle regulation in the course of nodule organogenesis in Medicago.

The molecular mechanisms of de novo meristem formation, cell differentiation and the integration of the cell cycle machinery into appropriate stages of the developmental programmes are still largely unknown in plants. Legume root nodules, which house nitrogen-fixing rhizobia, are unique plant organs and their development may serve as a model for organogenetic processes in plants. Nodules form and are essential for the plant only under limitation of combined nitrogen in the soil. Moreover, their development is triggered by external mitogenic signals produced by their symbiotic partners, the rhizobia. These signals, the lipochitooligosaccharide Nod factors, act as host-specific morphogens and induce the re-entry of root cortical cells into mitotic cycles. Maintenance of cell division activity leads to the formation of a persistent nodule meristem from which cells exit continuously and enter the nodule differentiation programme, involving multiple cycles of endoreduplication and enlargement of nuclear and cell volumes. While the small diploid 2C cells remain uninfected, the large polyploid cells can be invaded and, after completing the differentiation programme, host the nitrogen-fixing bacteroids. This review summarizes the present knowledge on cell cycle reactivation and meristem formation in response to Nod factors and reports on a novel plant cell cycle regulator that can switch mitotic cycles to differentiation programmes.

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.

Succinoglycan is required for initiation and elongation of infection threads during nodulation of alfalfa by Rhizobium meliloti.

Rhizobium meliloti Rm1021 must be able to synthesize succinoglycan in order to invade successfully the nodules which it elicits on alfalfa and to establish an effective nitrogen-fixing symbiosis. Using R. meliloti cells that express green fluorescent protein (GFP), we have examined the nature of the symbiotic deficiency of exo mutants that are defective or altered in succinoglycan production. Our observations indicate that an exoY mutant, which does not produce succinoglycan, is symbiotically defective because it cannot initiate the formation of infection threads. An exoZ mutant, which produces succinoglycan without the acetyl modification, forms nitrogen-fixing nodules on plants, but it exhibits a reduced efficiency in the initiation and elongation of infection threads. An exoH mutant, which produces symbiotically nonfunctional high-molecular-weight succinoglycan that lacks the succinyl modification, cannot form extended infection threads. Infection threads initiate at a reduced rate and then abort before they reach the base of the root hairs. Overproduction of succinoglycan by the exoS96::Tn5 mutant does not reduce the efficiency of infection thread initiation and elongation, but it does significantly reduce the ability of this mutant to colonize the curled root hairs, which is the first step of the invasion process. The exoR95::Tn5 mutant, which overproduces succinoglycan to an even greater extent than the exoS96::Tn5 mutant, has completely lost its ability to colonize the curled root hairs. These new observations lead us to propose that succinoglycan is required for both the initiation and elongation of infection threads during nodule invasion and that excess production of succinoglycan interferes with the ability of the rhizobia to colonize curled root hairs.

Induction of microbial genes for pathogenesis and symbiosis by chemicals from root border cells.

Reporter strains of soil-borne bacteria were used to test the hypothesis that chemicals released by root border cells can influence the expression of bacterial genes required for the establishment of plant-microbe associations. Promoters from genes known to be activated by plant factors included virE, required for Agrobacterium tumefaciens pathogenesis, and common nod genes from Rhizobium leguminosarum bv viciae and Rhizobium meliloti, required for nodulation of pea (Pisum sativum) and alfalfa (Medicago sativum), respectively. Also included was phzB, an autoinducible gene encoding the biosynthesis of antibiotics by Pseudomonas aureofaciens. The virE and nod genes were activated to different degrees, depending on the source of border cells, whereas phzB activity remained unaffected. The homologous interaction between R. leguminosarum bv viciae and its host, pea, was examined in detail. Nod gene induction by border cells was dosage dependent and responsive to environmental signals. The highest levels of gene induction by pea (but not alfalfa) border cells occurred at low temperatures, when little or no bacterial growth was detected. Detached border cells cultured in distilled water exhibited increased nod gene induction (ini) in response to signals from R. leguminosarum bv viciae.

Transient induction of a peroxidase gene in Medicago truncatula precedes infection by Rhizobium meliloti.

Although key determinative events of the Rhizobium-legume symbiosis are likely to precede bacterial infection, no plant genes have been identified that are expressed strongly prior to infection and nodule morphogenesis. A subtractive hybridization-polymerase chain reaction technique was used to enrich for genes induced during the early phases of the R. meliloti-Medicago truncatula symbiosis. One gene so identified encodes a putative plant peroxidase protein, which we have named Rip1 for Rhizobium-induced peroxidase. The accumulation of rip1 transcript was rapidly and transiently induced by R. meliloti and by the corresponding lipooligosaccharide signal molecule Nod factor RmIV, which was both necessary and sufficient for rip1 induction. The duration of maximal rip1 expression coincided with the preinfection period: transcript levels for rip1 were near maximal by 3 hr postinoculation and declined by 48 hr, coincident with early infection events and the onset of nodule morphogenesis. Furthermore, although rip1 induction preceded bacterial infection by at least 24 hr, the transcript was localized to epidermal cells in the differentiating root zone that was subsequently infected by Rhizobium. Thus, a defining feature of the Rhizobium infection court is the prior induction of rip1 expression.

The acetyl substituent of succinoglycan is not necessary for alfalfa nodule invasion by Rhizobium meliloti Rm1021.

Rhizobium meliloti Rm1021 requires a Calcofluor-binding exopolysaccharide, termed succinoglycan or EPS I, to invade alfalfa nodules. We have determined that a strain carrying a mutation in the exoZ locus produces succinoglycan that lacks the acetyl substituent. The exoZ mutant nodules alfalfa normally.