Differential Stem Proteomics and Metabolomics Profiles for Four Wheat Cultivars in Response to the Insect Pest Wheat Stem Sawfly.

Common wheat (Triticum aestivum L.) is a global staple crop, and insect pests can impact grain yield. The wheat stem sawfly (Cephus cinctus, WSS) is a major wheat pest, and while partial resistance has been deployed by breeding for a solid-stem trait, this trait is affected by environment. Here, a proteomics and metabolomics study was performed on four wheat cultivars to characterize a molecular response to WSS infestation. The cultivars Hatcher (hollow-stem partially tolerant), Conan (semisolid-stem-resistant), and Denali and Reeder (hollow-stem-susceptible) were infested with WSS, and changes in stem proteins and metabolites were characterized using liquid chromatography-mass spectrometry. The proteome was characterized as 1830 proteins that included five major biological processes, including metabolic processes and response to stimuli, and the metabolome (1823 metabolites) spanned eight chemical superclasses, including alkaloids, benzenoids, and lipids. All four varieties had a molecular response to WSS following infestation. Hatcher had the most distinct changes, whereby 62 proteins and 29 metabolites varied in metabolic pathways involving enzymatic detoxification, proteinase inhibition, and antiherbivory compound production via benzoxazinoids, neolignans, and phenolics. Taken together, these data demonstrate variation in the wheat stem molecular response to WSS infestation and support breeding for molecular resistance in hollow-stem cultivars.

A Program for Iron Economy during Deficiency Targets Specific Fe Proteins.

Iron (Fe) is an essential element for plants, utilized in nearly every cellular process. Because the adjustment of uptake under Fe limitation cannot satisfy all demands, plants need to acclimate their physiology and biochemistry, especially in their chloroplasts, which have a high demand for Fe. To investigate if a program exists for the utilization of Fe under deficiency, we analyzed how hydroponically grown Arabidopsis (Arabidopsis thaliana) adjusts its physiology and Fe protein composition in vegetative photosynthetic tissue during Fe deficiency. Fe deficiency first affected photosynthetic electron transport with concomitant reductions in carbon assimilation and biomass production when effects on respiration were not yet significant. Photosynthetic electron transport function and protein levels of Fe-dependent enzymes were fully recovered upon Fe resupply, indicating that the Fe depletion stress did not cause irreversible secondary damage. At the protein level, ferredoxin, the cytochrome-b6f complex, and Fe-containing enzymes of the plastid sulfur assimilation pathway were major targets of Fe deficiency, whereas other Fe-dependent functions were relatively less affected. In coordination, SufA and SufB, two proteins of the plastid Fe-sulfur cofactor assembly pathway, were also diminished early by Fe depletion. Iron depletion reduced mRNA levels for the majority of the affected proteins, indicating that loss of enzyme was not just due to lack of Fe cofactors. SufB and ferredoxin were early targets of transcript down-regulation. The data reveal a hierarchy for Fe utilization in photosynthetic tissue and indicate that a program is in place to acclimate to impending Fe deficiency.

High-throughput quantitative analysis of phytohormones in sorghum leaf and root tissue by ultra-performance liquid chromatography-mass spectrometry.

Plant development, growth, and adaptation to stress are regulated by phytohormones, which can influence physiology even at low concentrations. Phytohormones are chemically grouped according to both structure and function as auxins, cytokinins, abscisic acid, jasmonates, salicylates, gibberellins, and brassinosteroids, among others. This chemical diversity and requirement for highly sensitive detection in complex matrices create unique challenges for comprehensive phytohormone analysis. Here, we present a robust and efficient quantitative UPLC-MS/MS assay for 17 phytohormones, including jasmonates, salicylates, abscisic acid, gibberellins, cytokinins, and auxins. Using this assay, 12 phytohormones were detected and quantified in sorghum plant tissue without the need for solid phase extraction (SPE) or liquid-liquid extraction. Variation of phytohormone profiles was explored in both root and leaf tissues between three genotypes, harvested at two different developmental time points. The results highlight the importance of tissue type, sampling time, and genetic factors when designing experiments that involve phytohormone analysis of sorghum. This research lays the groundwork for future studies, which can combine phytohormone profiling with other datasets such as transcriptome, soil microbiome, genome, and metabolome data, to provide important functional information about adaptation to stress and other environmental variables.

Non-Targeted Metabolomics Reveals Sorghum Rhizosphere-Associated Exudates are Influenced by the Belowground Interaction of Substrate and Sorghum Genotype.

Root exudation is an important plant process by which roots release small molecules into the rhizosphere that serve in overall plant functioning. Yet, there is a major gap in our knowledge in translating plant root exudation in artificial systems (i.e., hydroponics, sterile media) to crops, specifically for soils expected in field conditions. Sorghum (Sorghum bicolor L. Moench) root exudation was determined using both ultra-performance liquid chromatography and gas chromatography mass spectrometry-based non-targeted metabolomics to evaluate variation in exudate composition of two sorghum genotypes among three substrates (sand, clay, and soil). Above and belowground plant traits were measured to determine the interaction between sorghum genotype and belowground substrate. Plant growth and quantitative exudate composition were found to vary largely by substrate. Two types of changes to rhizosphere metabolites were observed: rhizosphere-enhanced metabolites (REMs) and rhizosphere-abated metabolites (RAMs). More REMs and RAMs were detected in sand and clay substrates compared to the soil substrate. This study demonstrates that belowground substrate influences the root exudate profile in sorghum, and that two sorghum genotypes exuded metabolites at different magnitudes. However, metabolite identification remains a major bottleneck in non-targeted metabolite profiling of the rhizosphere.

Common bean varieties demonstrate differential physiological and metabolic responses to the pathogenic fungus Sclerotinia sclerotiorum.

Plant physiology and metabolism are important components of a plant response to microbial pathogens. Physiological resistance of common bean (Phaseolus vulgaris L.) to the fungal pathogen Sclerotinia sclerotiorum has been established, but the mechanisms of resistance are largely unknown. Here, the physiological and metabolic responses of bean varieties that differ in physiological resistance to S. sclerotiorum are investigated. Upon infection, the resistant bean variety A195 had a unique physiological response that included reduced photosynthesis and maintaining a higher leaf surface pH during infection. Leaf metabolomics was performed on healthy tissue adjacent to the necrotic lesion at 16, 24, and 48 hr post inoculation, and 144 metabolites were detected that varied between A195 and Sacramento following infection. The metabolites that varied in leaves included amines/amino acids, organic acids, phytoalexins, and ureides. The metabolic pathways associated with resistance included amine metabolism, uriede-based nitrogen remobilization, antioxidant production, and bean-specific phytoalexin production. A second experiment was conducted in stems of 13 bean genotypes with varying resistance. Stem resistance was associated with phytoalexin production, but unlike leaf metabolism, lipid changes were associated with susceptibility. Taken together, the data supports a multifaceted, physiometabolic response of common bean to S. sclerotiorum that mediates resistance.

GC-MS Metabolomics to Evaluate the Composition of Plant Cuticular Waxes for Four Triticum aestivum Cultivars.

Wheat (Triticum aestivum L.) is an important food crop, and biotic and abiotic stresses significantly impact grain yield. Wheat leaf and stem surface waxes are associated with traits of biological importance, including stress resistance. Past studies have characterized the composition of wheat cuticular waxes, however protocols can be relatively low-throughput and narrow in the range of metabolites detected. Here, gas chromatography-mass spectrometry (GC-MS) metabolomics methods were utilized to provide a comprehensive characterization of the chemical composition of cuticular waxes in wheat leaves and stems. Further, waxes from four wheat cultivars were assayed to evaluate the potential for GC-MS metabolomics to describe wax composition attributed to differences in wheat genotype. A total of 263 putative compounds were detected and included 58 wax compounds that can be classified (e.g., alkanes and fatty acids). Many of the detected wax metabolites have known associations to important biological functions. Principal component analysis and ANOVA were used to evaluate metabolite distribution, which was attributed to both tissue type (leaf, stem) and cultivar differences. Leaves contained more primary alcohols than stems such as 6-methylheptacosan-1-ol and octacosan-1-ol. The metabolite data were validated using scanning electron microscopy of epicuticular wax crystals which detected wax tubules and platelets. Conan was the only cultivar to display alcohol-associated platelet-shaped crystals on its abaxial leaf surface. Taken together, application of GC-MS metabolomics enabled the characterization of cuticular wax content in wheat tissues and provided relative quantitative comparisons among sample types, thus contributing to the understanding of wax composition associated with important phenotypic traits in a major crop.

CYCD3 D-type cyclins regulate cambial cell proliferation and secondary growth in Arabidopsis.

A major proportion of plant biomass is derived from the activity of the cambium, a lateral meristem responsible for vascular tissue formation and radial organ enlargement in a process termed secondary growth. In contrast to our relatively good understanding of the regulation of primary meristems, remarkably little is known concerning the mechanisms controlling secondary growth, particularly how cambial cell divisions are regulated and integrated with vascular differentiation. A genetic loss-of-function approach was used here to reveal a rate-limiting role for the Arabidopsis CYCLIN D3 (CYCD3) subgroup of cell-cycle genes in the control of cambial cell proliferation and secondary growth, providing conclusive evidence of a direct link between the cell cycle and vascular development. It is shown that all three CYCD3 genes are specifically expressed in the cambium throughout vascular development. Analysis of a triple loss-of-function CYCD3 mutant revealed a requirement for CYCD3 in promoting the cambial cell cycle since mutant stems and hypocotyls showed a marked reduction in diameter linked to reduced mitotic activity in the cambium. Conversely, loss of CYCD3 provoked an increase in xylem cell size and the expression of differentiation markers, showing that CYCD3 is required to restrain the differentiation of xylem precursor cells. Together, our data show that tight control of cambial cell division through developmental- and cell type-specific regulation of CYCD3 is required for normal vascular development, constituting part of a novel mechanism controlling organ growth in higher plants.

Genetic variation in biomass traits among 20 diverse rice varieties.

Biofuels provide a promising route of producing energy while reducing reliance on petroleum. Developing sustainable liquid fuel production from cellulosic feedstock is a major challenge and will require significant breeding efforts to maximize plant biomass production. Our approach to elucidating genes and genetic pathways that can be targeted for improving biomass production is to exploit the combination of genomic tools and genetic diversity in rice (Oryza sativa). In this study, we analyzed a diverse set of 20 recently resequenced rice varieties for variation in biomass traits at several different developmental stages. The traits included plant size and architecture, aboveground biomass, and underlying physiological processes. We found significant genetic variation among the 20 lines in all morphological and physiological traits. Although heritability estimates were significant for all traits, heritabilities were higher in traits relating to plant size and architecture than for physiological traits. Trait variation was largely explained by variety and breeding history (advanced versus landrace) but not by varietal groupings (indica, japonica, and aus). In the context of cellulosic biofuels development, cell wall composition varied significantly among varieties. Surprisingly, photosynthetic rates among the varieties were inversely correlated with biomass accumulation. Examining these data in an evolutionary context reveals that rice varieties have achieved high biomass production via independent developmental and physiological pathways, suggesting that there are multiple targets for biomass improvement. Future efforts to identify loci and networks underlying this functional variation will facilitate the improvement of biomass traits in other grasses being developed as energy crops.

The Dickeya dadantii biofilm matrix consists of cellulose nanofibres, and is an emergent property dependent upon the type III secretion system and the cellulose synthesis operon.

Dickeya dadantii is a plant-pathogenic bacterium that produces cellulose-containing biofilms, called pellicles, at the air-liquid interface of liquid cultures. D. dadantii pellicle formation appears to be an emergent property dependent upon at least three gene clusters, including cellulose synthesis, type III secretion system (T3SS) and flagellar genes. The D. dadantii cellulose synthesis operon is homologous to that of Gluconacetobacter xylinus, which is used for industrial cellulose production, and the cellulose nanofibres produced by D. dadantii were similar in diameter and branching pattern to those produced by G. xylinus. Salmonella enterica, an enterobacterium closely related to D. dadantii, encodes a second type of cellulose synthesis operon, and it produced biofilm strands that differed in width and branching pattern from those of D. dadantii and G. xylinus. Unlike any previously described cellulose fibre, the D. dadantii cellulose nanofibres were decorated with bead-like structures. Mutation of the cellulose synthesis operon genes resulted in loss of cellulose synthesis and production of a cellulase-resistant biofilm. Mutation of other genes required for pellicle formation, including those encoding FliA (a sigma factor that regulates flagella production), HrpL (a sigma factor that regulates the T3SS), and AdrA, a GGDEF protein, affected both biofilm and cell morphology. Mutation of the cellulose synthase bcsA or of bcsC resulted in decreased accumulation of the T3SS-secreted protein HrpN.

Drought-Induced Root Pressure in Sorghum bicolor.

Root pressure, also manifested as profusive sap flowing from cut stems, is a phenomenon in some species that has perplexed biologists for much of the last century. It is associated with increased crop production under drought, but its function and regulation remain largely unknown. In this study, we investigated the initiation, mechanisms, and possible adaptive function of root pressure in six genotypes of Sorghum bicolor during a drought experiment in the greenhouse. We observed that root pressure was induced in plants exposed to drought followed by re-watering but possibly inhibited by 100% re-watering in some genotypes. We found that root pressure in drought stressed and re-watered plants was associated with greater ratio of fine: coarse root length and shoot biomass production, indicating a possible role of root allocation in creating root pressure and adaptive benefit of root pressure for shoot biomass production. Using RNA-Seq, we identified gene transcripts that were up- and down-regulated in plants with root pressure expression, focusing on genes for aquaporins, membrane transporters, and ATPases that could regulate inter- and intra-cellular transport of water and ions to generate positive xylem pressure in root tissue.

Interactions of free-living amoebae with the rice fungal pathogen, Rhizoctonia solani.

OBJECTIVE: Rhizoctonia solani is a soil-borne fungal pathogen of many important crop plants. In rice, R. solani causes sheath blight disease, which results in devastating grain yield and quality losses. Few methods are available to control this pathogen and classic single gene resistance mechanisms in rice plants have not been identified. We hypothesize that alternate means of control are available in the environment including free-living amoebae. Amoebae are soil-, water- and air-borne microorganisms that are predominantly heterotrophic. Many amoeba species are mycophagous, and several harm their prey using mechanisms other than phagocytosis. Here, we used light and scanning electron microscopy to survey the interactions of R. solani with four amoeba species, with the goal of identifying amoebae species with potential for biocontrol. RESULTS: We observed a wide range of responses during interactions of R. solani with four different free-living amoebae. Two Acanthamoeba species encyst in co-cultures with R. solani at higher rates than medium without R. solani. Vermamoeba vermiformis (formerly Hartmanella vermiformis) attach to R. solani mycelium and are associated with mycelial shriveling and perforations of fungal cell walls, indicating an antagonistic interaction. No phenotypic changes were observed in co-cultures of Dictyostelium discoideum and R. solani.

Potential for Beneficial Reuse of Oil and Gas-Derived Produced Water in Agriculture: Physiological and Morphological Responses in Spring Wheat (Triticum aestivum).

Produced water (PW) from oil and gas operations is considered a potential resource for food crop irrigation because of increasing water scarcity in dryland agriculture. However, efforts to employ PW for agriculture have been met with limited success. A greenhouse study was performed to evaluate the effects of PW on physiological and morphological traits of spring wheat (Triticum aestivum). Plants were irrigated with water treatments containing 10 and 50% PW (PW10 and PW50, respectively) and compared to a matching 50% salinity (NaCl50) and 100% tap water controls. Compared to controls, plants watered with PW10 and PW50 exhibited developmental arrest and reductions in aboveground and belowground biomass, photosynthetic efficiency, and reproductive growth. Decreases in grain yield ranged from 70 to 100% in plants irrigated with PW compared to the tap water control. Importantly, the PW10 and NaCl50 treatments were comparable for morphophysiological effects, even though NaCl50 contained 5 times the total dissolved solids, suggesting that constituents other than NaCl in PW contributed to plant stress. These findings indicate that despite discharge and reuse requirements focused on total dissolved solids, salinity stress may not be the primary factor affecting crop health. The results of the present study are informative for developing guidelines for the use of PW in agriculture to ensure minimal effects on crop morphology and physiology. Environ Toxicol Chem 2019;38:1756-1769. © 2019 SETAC.

The flagellar sigma factor fliA is required for Dickeya dadantii virulence.

The genome sequence of the Enterobacteriaceae phytopathogen Dickeya dadantii (formerly Erwinia chrysanthemi) revealed homologs of genes required for a complete flagellar secretion system and one flagellin gene. We found that D. dadantii was able to swim and swarm but that ability to swarm was dependent upon both growth media and temperature. Mutation of the D. dadantii fliA gene was pleiotropic, with the alternate sigma factor required for flagella production and development of disease symptoms but not bacterial growth in Nicotiana benthamiana leaves. The flagellar sigma factor was also required for multiple bacterial phenotypes, including biofilm formation in culture, bacterial adherence to plant tissue, and full expression of pectate lyase activity (but not cellulase or protease activity). Surprisingly, mutation of fliA resulted in the increased expression of avrL (a gene of unknown function in D. dadantii) and two pectate lyase gene homologs, pelX and ABF-0019391. Because FliA is a key contributor to virulence in D. dadantii, it is a new target for disease control.

Evaluation of isolation methods and RNA integrity for bacterial RNA quantitation.

RNA integrity is critical for successful RNA quantitation for mammalian tissues, but the level of integrity required differs among tissues. The level of integrity required for quantitation has not been determined for bacterial RNA. Three RNA isolation methods were evaluated for their ability to produce high quality RNA from Dickeya dadantii, a bacterium refractory to RNA isolation. Bacterial lysis with Trizol using standard protocols consistently gave low RNA yields with this organism. Higher yields due to improved bacterial cells lysis was achieved with an added hot SDS incubation step, but RNA quality was low as determined by the RNA Integrity Number (RIN). Contaminating DNA remained a problem with the hot SDS-Trizol method; RNA samples required repeated, rigorous DNase treatments to reduce DNA contamination to levels sufficient for successful real-time qRT-PCR. A hot SDS-hot phenol RNA method gave the highest RNA quality and required only two DNase treatments to remove DNA. The assessment of RNA integrity using the Agilent 2100 BioAnalyzer was critical for obtaining meaningful gene expression data. RIN values below 7.0 resulted in high variation and loss of statistical significance when gene expression was analyzed by real-time qRT-PCR. We found that RNA preparations of different quality yielded drastic differences in relative gene expression ratios and led to major errors in the quantification of transcript levels. This work provides guidelines for RNA isolation and quality assessment that will be valuable for gene expression studies in a wide range of bacteria.

Interactions of free-living amoebae with rice bacterial pathogens Xanthomonas oryzae pathovars oryzae and oryzicola.

BACKGROUND: Free-living amoebae (FLA) are voracious feeders, consuming bacteria and other microbes during colonization of the phytobiome. FLA are also known to secrete bacteriocidal or bacteriostatic compounds into their growth environment. METHODOLOGY AND PRINCIPAL FINDINGS: Here, we explore the impacts of co-cultivation of five FLA species, including Acanthamoeba castellanii, A. lenticulata, A. polyphaga, Dictyostelium discoideum and Vermamoeba vermiformis, on survival of two devastating bacterial pathogens of rice, Xanthomonas oryzae pathovars (pv.) oryzae and oryzicola. In co-cultivation assays, the five FLA species were either bacteriostatic or bactericidal to X. oryzae pv. oryzae and X. oryzae pv. oryzicola. Despite these effects, bacteria were rarely detected inside amoebal cells. Furthermore, amoebae did not disrupt X. oryzae biofilms. The bactericidal effects persisted when bacteria were added to a cell-free supernatant from amoebal cultures, suggesting some amoebae produce an extracellular bactericidal compound. CONCLUSIONS/SIGNIFICANCE: This work establishes novel, basal dynamics between important plant pathogenic bacteria and diverse amoebae, and lays the framework for future mechanistic studies.

The role of cellulose and O-antigen capsule in the colonization of plants by Salmonella enterica.

Numerous salmonellosis outbreaks have been associated with vegetables, in particular sprouted seed. Thin aggregative fimbriae (Tafi), a component of the extracellular matrix responsible for multicellular behavior, are important for Salmonella enterica attachment and colonization of plants. Here, we demonstrate that the other surface polymers composing the extracellular matrix, cellulose, and O-antigen capsule also play a role in colonization of plants. Mutations in bacterial cellulose synthesis (bcsA) and O-antigen capsule assembly and translocation (yihO) reduced the ability to attach to and colonize alfalfa sprouts. A colanic acid mutant was unaffected in plant attachment or colonization. Tafi, cellulose synthesis, and O-antigen capsule, all of which contribute to attachment and colonization of plants, are regulated by AgfD, suggesting that AgfD is a key regulator for survival outside of hosts of Salmonella spp. The cellulose biosynthesis regulator adrA mutant was not affected in the ability to attach to or colonize plants; however, promoter probe assays revealed expression by cells attached to alfalfa sprouts. Furthermore, quantitative reverse-transcriptase polymerase chain reaction revealed differential expression of agfD and adrA between planktonic and plant-attached cells. In addition, there was no correlation among mutants between biofilm formation in culture and attachment to plants. Outside of animal hosts, S. enterica appears to rely on an arsenal of adhesins to persist on plants, which can act as vectors and perpetuate public health concerns.

Non-targeted Metabolomics in Diverse Sorghum Breeding Lines Indicates Primary and Secondary Metabolite Profiles Are Associated with Plant Biomass Accumulation and Photosynthesis.

Metabolomics is an emerging method to improve our understanding of how genetic diversity affects phenotypic variation in plants. Recent studies have demonstrated that genotype has a major influence on biochemical variation in several types of plant tissues, however, the association between metabolic variation and variation in morphological and physiological traits is largely unknown. Sorghum bicolor (L.) is an important food and fuel crop with extensive genetic and phenotypic variation. Sorghum lines have been bred for differing phenotypes beneficial for production of grain (food), stem sugar (food, fuel), and cellulosic biomass (forage, fuel), and these varying phenotypes are the end products of innate metabolic programming which determines how carbon is allocated during plant growth and development. Further, sorghum has been adapted among highly diverse environments. Because of this geographic and phenotypic variation, the sorghum metabolome is expected to be highly divergent; however, metabolite variation in sorghum has not been characterized. Here, we utilize a phenotypically diverse panel of sorghum breeding lines to identify associations between leaf metabolites and morpho-physiological traits. The panel (11 lines) exhibited significant variation for 21 morpho-physiological traits, as well as broader trends in variation by sorghum type (grain vs. biomass types). Variation was also observed for cell wall constituents (glucan, xylan, lignin, ash). Non-targeted metabolomics analysis of leaf tissue showed that 956 of 1181 metabolites varied among the lines (81%, ANOVA, FDR adjusted p < 0.05). Both univariate and multivariate analyses determined relationships between metabolites and morpho-physiological traits, and 384 metabolites correlated with at least one trait (32%, p < 0.05), including many secondary metabolites such as glycosylated flavonoids and chlorogenic acids. The use of metabolomics to explain relationships between two or more morpho-physiological traits was explored and showed chlorogenic and shikimic acid to be associated with photosynthesis, early plant growth and final biomass measures in sorghum. Taken together, this study demonstrates the integration of metabolomics with morpho-physiological datasets to elucidate links between plant metabolism, growth, and architecture.

Biomass for thermochemical conversion: targets and challenges.

Bioenergy will be one component of a suite of alternatives to fossil fuels. Effective conversion of biomass to energy will require the careful pairing of advanced conversion technologies with biomass feedstocks optimized for the purpose. Lignocellulosic biomass can be converted to useful energy products via two distinct pathways: enzymatic or thermochemical conversion. The thermochemical pathways are reviewed and potential biotechnology or breeding targets to improve feedstocks for pyrolysis, gasification, and combustion are identified. Biomass traits influencing the effectiveness of the thermochemical process (cell wall composition, mineral and moisture content) differ from those important for enzymatic conversion and so properties are discussed in the language of biologists (biochemical analysis) as well as that of engineers (proximate and ultimate analysis). We discuss the genetic control, potential environmental influence, and consequences of modification of these traits. Improving feedstocks for thermochemical conversion can be accomplished by the optimization of lignin levels, and the reduction of ash and moisture content. We suggest that ultimate analysis and associated properties such as H:C, O:C, and heating value might be more amenable than traditional biochemical analysis to the high-throughput necessary for the phenotyping of large plant populations. Expanding our knowledge of these biomass traits will play a critical role in the utilization of biomass for energy production globally, and add to our understanding of how plants tailor their composition with their environment.

The Erwinia chrysanthemi type III secretion system is required for multicellular behavior.

Enterobacterial animal pathogens exhibit aggregative multicellular behavior, which is manifested as pellicles on the culture surface and biofilms at the surface-liquid-air interface. Pellicle formation behavior requires production of extracellular polysaccharide, cellulose, and protein filaments, known as curli. Protein filaments analogous to curli are formed by many protein secretion systems, including the type III secretion system (TTSS). Here, we demonstrate that Erwinia chrysanthemi, which does not carry curli genes, requires the TTSS for pellicle formation. These data support a model where cellulose and generic protein filaments, which consist of either curli or TTSS-secreted proteins, are required for enterobacterial aggregative multicellular behavior. Using this assay, we found that hrpY, which encodes a two-component system response regulator homolog, is required for activity of hrpS, which encodes a sigma54-dependent enhancer-binding protein homolog. In turn, hrpS is required for activity of the sigma factor homolog hrpL, which activates genes encoding TTSS structural and secreted proteins. Pellicle formation was temperature dependent and pellicles did not form at 36 degrees C, even though TTSS genes were expressed at this temperature. We found that cellulose is a component of the E. chrysanthemi pellicle but that pellicle formation still occurs in a strain with an insertion in a cellulose synthase subunit homolog. Since the TTSS, but not the cellulose synthase subunit, is required for E. chrysanthemi pellicle formation, this inexpensive assay can be used as a high throughput screen for TTSS mutants or inhibitors.

Cultivation of mesophilic soil crenarchaeotes in enrichment cultures from plant roots.

Because archaea are generally associated with extreme environments, detection of nonthermophilic members belonging to the archaeal division Crenarchaeota over the last decade was unexpected; they are surprisingly ubiquitous and abundant in nonextreme marine and terrestrial habitats. Metabolic characterization of these nonthermophilic crenarchaeotes has been impeded by their intractability toward isolation and growth in culture. From studies employing a combination of cultivation and molecular phylogenetic techniques (PCR-single-strand conformation polymorphism, sequence analysis of 16S rRNA genes, fluorescence in situ hybridization, and real-time PCR), we present evidence here that one of the two dominant phylotypes of Crenarchaeota that colonizes the roots of tomato plants grown in soil from a Wisconsin field is selectively enriched in mixed cultures amended with root extract. Clones recovered from enrichment cultures were found to group phylogenetically with sequences from clade C1b.A1. This work corroborates and extends our recent findings, indicating that the diversity of the crenarchaeal soil assemblage is influenced by the rhizosphere and that mesophilic soil crenarchaeotes are found associated with plant roots, and provides the first evidence for growth of nonthermophilic crenarchaeotes in culture.