Spatially Restricted Immune Responses Are Required for Maintaining Root Meristematic Activity upon Detection of Bacteria.

Plants restrict immune responses to vulnerable root parts. Spatially restricted responses are thought to be necessary to avoid constitutive responses to rhizosphere microbiota. To directly demonstrate the importance of spatially restricted responses, we expressed the plant flagellin receptor (FLS2) in different tissues, combined with fluorescent defense markers for immune readouts at cellular resolution. Our analysis distinguishes responses appearing cell autonomous from apparently non-cell-autonomous responses. It reveals lignification as a general immune response, contrasting suberization. Importantly, our analysis divides the root meristem into a central zone refractory to FLS2 expression and a cortex that is sensitized by FLS2 expression, causing meristem collapse upon stimulation. Meristematic epidermal expression generates super-competent lines that detect native bacterial flagellin and bypass the weak or absent response to commensals, providing a powerful tool for studying root immunity. Our manipulations and readouts demonstrate incompatibility of meristematic activity and defense and the importance of cell-resolved studies of plant immune responses.

The plant beneficial rhizobacterium Achromobacter sp. 5B1 influences root development through auxin signaling and redistribution.

Roots provide physical and nutritional support to plant organs that are above ground and play critical roles for adaptation via intricate movements and growth patterns. Through screening the effects of bacterial isolates from roots of halophyte Mesquite (Prosopis sp.) on Arabidopsis thaliana, we identified Achromobacter sp. 5B1 as a probiotic bacterium that influences plant functional traits. Detailed genetic and architectural analyses in Arabidopsis grown in vitro and in soil, cell division measurements, auxin transport and response gene expression and brefeldin A treatments demonstrated that root colonization with Achromobacter sp. 5B1 changes the growth and branching patterns of roots, which were related to auxin perception and redistribution. Expression analysis of auxin transport and signaling revealed a redistribution of auxin within the primary root tip of wild-type seedlings by Achromobacter sp. 5B1 that is disrupted by brefeldin A and correlates with repression of auxin transporters PIN1 and PIN7 in root provasculature, and PIN2 in the epidermis and cortex of the root tip, whereas expression of PIN3 was enhanced in the columella. In seedlings harboring AUX1, EIR1, AXR1, ARF7ARF19, TIR1AFB2AFB3 single, double or triple loss-of-function mutations, or in a dominant (gain-of-function) mutant of SLR1, the bacterium caused primary roots to form supercoils that are devoid of lateral roots. The changes in growth and root architecture elicited by the bacterium helped Arabidopsis seedlings to resist salt stress better. Thus, Achromobacter sp. 5B1 fine tunes both root movements and the auxin response, which may be important for plant growth and environmental adaptation.

Plant tumors: a hundred years of study.

MAIN CONCLUSION: The review provides information on the mechanisms underlying the development of spontaneous and pathogen-induced tumors in higher plants. The activation of meristem-specific regulators in plant tumors of various origins suggests the meristem-like nature of abnormal plant hyperplasia. Plant tumor formation has more than a century of research history. The study of this phenomenon has led to a number of important discoveries, including the development of the Agrobacterium-mediated transformation technique and the discovery of horizontal gene transfer from bacteria to plants. There are two main groups of plant tumors: pathogen-induced tumors (e.g., tumors induced by bacteria, viruses, fungi, insects, etc.), and spontaneous ones, which are formed in the absence of any pathogen in plants with certain genotypes (e.g., interspecific hybrids, inbred lines, and mutants). The causes of the transition of plant cells to tumor growth are different from those in animals, and they include the disturbance of phytohormonal balance and the acquisition of meristematic characteristics by differentiated cells. The aim of this review is to discuss the mechanisms underlying the development of most known examples of plant tumors.

Bacterial Shoot Apical Meristem Inoculation Assay.

By virtue of their sessile nature, plants may not show the fight-and-flight response, but they are not devoid of protecting themselves from disease-causing agents, attack by herbivores, and damages that are caused by other environmental factors. Plants differentially protect their life-sustaining organs such as plant apexes from the attack by microbial pathogens. There are well-established methods to inoculate/infect various plant parts such as leaves, roots, and stems with various different pathogens. The plant shoot apical meristems (SAM) are a high-value plant target that provides niche to stem cell populations. These stem cells are instrumental in maintaining future plant progenies by giving birth to cells that culminate in flowers, leaves, and stems. There are hardly few protocols available that allow us to study immune dynamics of the plant stem cells as they are hindered by various layers of the SAM cell populations. Here, we describe a step-by-step method on how to inoculate the Arabidopsis SAM with model plant pathogen Pseudomonas syringae pv. tomato DC3000.

Plant-Associated Microbes Alter Root Growth by Modulating Root Apical Meristem.

Rhizobacteria are known to produce a variety of signal molecules which may modify plant growth by interfering with phytohormone balance. Among the microbial signals are phytohormones, known to contribute to plant endogenous pool of phytohormones. The current chapter describes different methods to study the regulation of gene expression in root apical meristem in response to rhizobacterial inoculation. We describe protocol for the detection of in planta modulation of CKs and IAA by rhizobacteria and their impact on root growth, dissecting the underlying plant signaling pathway by RNA sequencing.

Flagellin of polar flagellum from Azospirillum brasilense Sp245: Isolation, structure, and biological activity.

Glycosylated flagellin of the polar flagellum of the plant growth-promoting rhizobacteria Azospirillum brasilense Sp245 was for the first time isolated and characterized by biochemical and bioinformatics methods. Using the amino acid sequence taken from the NCBI database of bacterial whole-genome DNA sequencing, the secondary and tertiary structures of the protein part of this glycoprotein were determined by template-based molecular modeling. With the use of a set of predictors, regions of its intrinsic structural disorder were identified, and binding sites of carbohydrate fragments to the surface of the molecule were determined. A positive effect of the polar flagellum flagellin on the root meristem of wheat seedlings was for the first time revealed for associative bacteria. The effect was manifested in an increase in the division rate of plant cells - a significant increase in the mitotic index. Thus, the induction of specific responses of plants to their interactions with flagellin of the associative bacteria may probably be considered as a demonstration of its elicitor properties.

Differential soil fungus accumulation and density dependence of trees in a subtropical forest.

The mechanisms underlying interspecific variation in conspecific negative density dependence (CNDD) are poorly understood. Using a multilevel modeling approach, we combined long-term seedling demographic data from a subtropical forest plot with soil fungal community data by means of DNA sequencing to address the feedback of various guilds of soil fungi on the density dependence of trees. We show that mycorrhizal type mediates tree neighborhood interactions at the community level, and much of the interspecific variation in CNDD is explained by how tree species differ in their fungal density accumulation rates as they grow. Species with higher accumulation rates of pathogenic fungi suffered more from CNDD, whereas species with lower CNDD had higher accumulation rates of ectomycorrhizal fungi, suggesting that mutualistic and pathogenic fungi play important but opposing roles.

New Symptoms Identified in Phytoplasma-Infected Plants Reveal Extra Stages of Pathogen-Induced Meristem Fate-Derailment.

In flowering plants, the transition of a shoot apical meristem from vegetative to reproductive destiny is a graduated, multistage process that involves sequential conversion of the vegetative meristem to an inflorescence meristem, initiation of floral meristems, emergence of flower organ primordia, and formation of floral organs. This orderly process can be derailed by phytoplasma, a bacterium that parasitizes phloem sieve cells. In a previous study, we showed that phytoplasma-induced malformation of flowers reflects stage-specific derailment of shoot apical meristems from their genetically preprogrammed reproductive destiny. Our current study unveiled new symptoms of abnormal morphogenesis, pointing to derailment of meristem transition at additional stages previously unidentified. We also found that the fate of developing meristems may be derailed even after normal termination of the floral meristem and onset of seed production. Although previous reports by others have indicated that different symptoms may be induced by different phytoplasmal effectors, the phenomenon observed in our experiment raises interesting questions as to (i) whether effectors can act at specific stages of meristem transition and (ii) whether specific floral abnormalities are attributable to meristem fate-derailment events triggered by different effectors that each act at a specific stage in meristem transition. Research addressing such questions may lead to discoveries of an array of phytoplasmal effectors.

Different endophyte communities colonize buds of sprouts compared with mature trees of mountain birch recovered from moth herbivory.

Plant meristems were previously thought to be sterile. Today, meristem-associated shoot endophytes are mainly reported as contaminants from plant tissue cultures, the number of observed species being very low. However, the few strains characterized have the capacity for infecting host cells and affecting plant growth and development. Here we studied the communities of endophytic bacteria in the buds of mountain birch (Betula pubescens ssp. czerepanovii (N. I. Orlova) Hämet-Ahti) exposed to winter moth (Operophtera brumata L.) herbivory, to identify differences between sprouts and branches of mature birch trees. Mountain birch of the high subarctic is cyclically exposed to winter moth and produces sprouts to generate new trees as a survival mechanism. The majority (54%) of operational taxonomic units belonged to Xanthomonadaceae and Pseudomonales of Proteobacteria. Most of the observed species were classified as Xanthomonas (28%). Sprout buds had the highest diversity, containing approximately three times more species, and significantly more (43%) Pseudomonas species than the mature trees (14%). Our results demonstrate that endophytic communities of buds are richer than previously thought. We suggest that the meristem-associated endophytes should be studied further for a possible role in sprouting and aiding regeneration of trees.

Susceptibility of Hop Crown Buds to Powdery Mildew and its Relation to Perennation of Podosphaera macularis.

In the Pacific Northwestern United States, the hop powdery mildew fungus, Podosphaera macularis, survives overwintering periods in association with living host tissue because the ascigerious stage of the pathogen is not known to occur in this region. Field experiments were conducted over a 5-year period to describe the overwintering process associated with crown bud infection and persistence of P. macularis. Surface crown buds increased in abundance and size beginning in early July and continuing until mid-September. Buds of varying sizes remained susceptible to powdery mildew until late September to early October in each of 3 years of experiments, with susceptibility decreasing substantially thereafter. Potted plants were inoculated sequentially during early summer to autumn, then evaluated in the following year for development of shoots colonized by the powdery mildew fungus (termed flag shoots) due to bud perennation. Emergence of flag shoots was asynchronous and associated with shoot emergence and elongation. Flag shoots emerged over a protracted period from late February to early June, year dependent. In all 4 years of experiments, some infected buds broke and produced flag shoots after chemical desiccation of shoots in spring, a common horticultural practice in hop production conducted to set training timing and eliminate initial inoculum. Flag shoots were most numerous when plants were inoculated with P. macularis in early summer and, consequently, when powdery mildew was present throughout the entire period of crown bud development. The number of flag shoots produced was reduced from 6.8- to 46.6-fold when comparing the latest versus earliest inoculation dates. However, all inoculation timings yielded flag shoots at some level, suggesting that bud infection that occurs over an extended period of time in the previous season may allow the fungus to perennate. In studies in two commercial hop yards in Washington State, fungicide applications made after harvest reduced the level of powdery mildew on leaves in the current year but did not significantly reduce flag shoots in the following year. Given that bud infection occurred over a 10-week period, flag shoots developed even when plants were exposed to inoculum in October and some flag shoots survived chemical pruning practices, management efforts seem best directed to both preventative measures to reduce the likelihood of bud infection and remedial practices to physically eliminate infected crown buds in the ensuing year.

Cacao Phylloplane: The First Battlefield against Moniliophthora perniciosa, Which Causes Witches' Broom Disease.

The phylloplane is the first contact surface between Theobroma cacao and the fungus Moniliophthora perniciosa, which causes witches' broom disease (WBD). We evaluated the index of short glandular trichomes (SGT) in the cacao phylloplane and the effect of irrigation on the disease index of cacao genotypes with or without resistance to WBD, and identified proteins present in the phylloplane. The resistant genotype CCN51 and susceptible Catongo presented a mean index of 1,600 and 700 SGT cm-2, respectively. The disease index in plants under drip irrigation was reduced by approximately 30% compared with plants under sprinkler irrigation prior to inoculation. Leaf water wash (LWW) of the cacao inhibited the germination of spores by up to 98%. Proteins from the LWW of CCN51 were analyzed by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by tandem mass spectrometry. The gel showed 71 spots and identified a total of 42 proteins (28 from the plant and 14 from bacteria). Proteins related to defense and synthesis of defense metabolites and involved in nucleic acid metabolism were identified. The results support the hypothesis that the proteins and water-soluble compounds secreted to the cacao phylloplane participate in the defense against pathogens. They also suggest that SGT can contribute to the resistance of cacao.

First detection of Endogone ectomycorrhizas in natural oak forests.

The order Endogonales in Mucoromycotina, an early divergent lineage of fungi, includes ectomycorrhizal (EM) fungi. This order is therefore considered a key taxon for elucidation of the evolution of EM associations. Recent studies have revealed high diversity of EM lineages of Basidiomycota and Ascomycota; however, EM associations of Endogonales and its relatives remain largely unknown. In this study, EM root tips with a unique fungal sheath, with aseptate and highly branched hyphae of variable widths, were identified in Quercus acutissima and Quercus crispula forests in the temperate zone of Japan. The mycobionts were confirmed as Endogone sp., which were placed as a sister clade of Endogone pisiformis, based on phylogenetic analyses of the small and large subunits of the nuclear ribosomal RNA and elongation factor-1α genes. This is the first report of EM of Endogone in natural forests of the Northern Hemisphere and the first finding on Quercus.

Transfer of 13 C between paired Douglas-fir seedlings reveals plant kinship effects and uptake of exudates by ectomycorrhizas.

Processes governing the fixation, partitioning, and mineralization of carbon in soils are under increasing scrutiny as we develop a more comprehensive understanding of global carbon cycling. Here we examined fixation by Douglas-fir seedlings and transfer to associated ectomycorrhizal fungi, soil microbes, and full-sibling or nonsibling neighbouring seedlings. Stable isotope probing with 99% 13 C-CO2 was applied to trace 13 C-labelled photosynthate throughout plants, fungi, and soil microbes in an experiment designed to assess the effect of relatedness on 13 C transfer between plant pairs. The fixation and transfer of the 13 C label to plant, fungal, and soil microbial tissue was examined in biomass and phospholipid fatty acids. After a 6 d chase period, c. 26.8% of the 13 C remaining in the system was translocated below ground. Enrichment was proportionally greatest in ectomycorrhizal biomass. The presence of mesh barriers (0.5 or 35 µm) between seedlings did not restrict 13 C transfer. Fungi were the primary recipients of 13 C-labelled photosynthate throughout the system, representing 60-70% of total 13 C-enriched phospholipids. Full-sibling pairs exhibited significantly greater 13 C transfer to recipient roots in two of four Douglas-fir families, representing three- and fourfold increases (+ c. 4 µg excess 13 C) compared with nonsibling pairs. The existence of a root/mycorrhizal exudation-hyphal uptake pathway was supported.

Root Border Cells and Their Role in Plant Defense.

Root border cells separate from plant root tips and disperse into the soil environment. In most species, each root tip can produce thousands of metabolically active cells daily, with specialized patterns of gene expression. Their function has been an enduring mystery. Recent studies suggest that border cells operate in a manner similar to mammalian neutrophils: Both cell types export a complex of extracellular DNA (exDNA) and antimicrobial proteins that neutralize threats by trapping pathogens and thereby preventing invasion of host tissues. Extracellular DNases (exDNases) of pathogens promote virulence and systemic spread of the microbes. In plants, adding DNase I to root tips eliminates border cell extracellular traps and abolishes root tip resistance to infection. Mutation of genes encoding exDNase activity in plant-pathogenic bacteria (Ralstonia solanacearum) and fungi (Cochliobolus heterostrophus) results in reduced virulence. The study of exDNase activities in plant pathogens may yield new targets for disease control.

Host specificity in Sporisorium reilianum is determined by distinct mechanisms in maize and sorghum.

Smut fungi are biotrophic plant pathogens that exhibit a very narrow host range. The smut fungus Sporisorium reilianum exists in two host-adapted formae speciales: S. reilianum f. sp. reilianum (SRS), which causes head smut of sorghum, and S. reilianum f. sp. zeae (SRZ), which induces disease on maize. It is unknown why the two formae speciales cannot form spores on their respective non-favoured hosts. By fungal DNA quantification and fluorescence microscopy of stained plant samples, we followed the colonization behaviour of both SRS and SRZ on sorghum and maize. Both formae speciales were able to penetrate and multiply in the leaves of both hosts. In sorghum, the hyphae of SRS reached the apical meristems, whereas the hyphae of SRZ did not. SRZ strongly induced several defence responses in sorghum, such as the generation of H2 O2 , callose and phytoalexins, whereas the hyphae of SRS did not. In maize, both SRS and SRZ were able to spread through the plant to the apical meristem. Transcriptome analysis of colonized maize leaves revealed more genes induced by SRZ than by SRS, with many of them being involved in defence responses. Amongst the maize genes specifically induced by SRS were 11 pentatricopeptide repeat proteins. Together with the microscopic analysis, these data indicate that SRZ succumbs to plant defence after sorghum penetration, whereas SRS proliferates in a relatively undisturbed manner, but non-efficiently, on maize. This shows that host specificity is determined by distinct mechanisms in sorghum and maize.

Ectomycorrhizal fungal assemblages of Abies alba Mill. outside its native range in Poland.

Abies alba (Mill.) is an important forest tree species, native to the mountainous regions of Europe but has been also widely introduced in the lowlands outside its native range. Like most forest tree species, A. alba forms obligate mutualisms with ectomycorrhizal (ECM) fungi. This investigation sought to examine ECM fungal communities of A. alba when the species grows 400 km north of its native range in the region of Pomerania in Poland. We surveyed for ECM fungi by sampling live roots from four mature forest stands where the A. alba component ranged from 20 to 100%. Ectomycorrhizal fungal symbionts were identified based on morphotyping and sequencing of the internal transcribed spacer (ITS) of nuclear ribosomal DNA (rDNA). Thirty-five ECM fungal taxa were distinguished on root tips of A. alba from all tested stands with 22 to 27 ECM fungal taxa in the individual stand. The diversity and similarity metrics revealed a lack of statistical differences in the structure of the ECM fungal community between stands varying in overstory tree composition. Cenococcum geophilum was the most common fungal species at all investigated A. alba stands, with an abundance of 50 to 70%. The ECM community was characterized by the lack of Abies-specific fungal symbionts and a rich and diverse suite of host-generalist mycobionts that seem to be sufficient for successful growth and development of A. alba outside of its native range.

Variation in ectomycorrhizal fungal communities associated with Oreomunnea mexicana (Juglandaceae) in a Neotropical montane forest.

Neotropical montane forests are often dominated by ectomycorrhizal (EM) tree species, yet the diversity of their EM fungal communities remains poorly explored. In lower montane forests in western Panama, the EM tree species Oreomunnea mexicana (Juglandaceae) forms locally dense populations in forest otherwise characterized by trees that form arbuscular mycorrhizal (AM) associations. The objective of this study was to compare the composition of EM fungal communities associated with Oreomunnea adults, saplings, and seedlings across sites differing in soil fertility and the amount and seasonality of rainfall. Analysis of fungal nrITS DNA (nuclear ribosomal internal transcribed spacers) revealed 115 EM fungi taxa from 234 EM root tips collected from adults, saplings, and seedlings in four sites. EM fungal communities were equally species-rich and diverse across Oreomunnea developmental stages and sites, regardless of soil conditions or rainfall patterns. However, ordination analysis revealed high compositional turnover between low and high fertility/rainfall sites located ca. 6 km apart. The EM fungal community was dominated by Russula (ca. 36 taxa). Cortinarius, represented by 14 species and previously reported to extract nitrogen from organic sources under low nitrogen availability, was found only in low fertility/high rainfall sites. Phylogenetic diversity analyses of Russula revealed greater evolutionary distance among taxa found on sites with contrasting fertility and rainfall than was expected by chance, suggesting that environmental differences among sites may be important in structuring EM fungal communities. More research is needed to evaluate whether EM fungal taxa associated with Oreomunnea form mycorrhizal networks that might account for local dominance of this tree species in otherwise diverse forest communities.

Interactive effects of juvenile defoliation, light conditions, and interspecific competition on growth and ectomycorrhizal colonization of Fagus sylvatica and Pinus sylvestris seedlings.

Seedlings of forest tree species are exposed to a number of abiotic (organ loss or damage, light shortage) and biotic (interspecific competition) stress factors, which may lead to an inhibition of growth and reproduction and, eventually, to plant death. Growth of the host and its mycorrhizal symbiont is often closely linked, and hence, host damage may negatively affect the symbiont. We designed a pot experiment to study the response of light-demanding Pinus sylvestris and shade-tolerant Fagus sylvatica seedlings to a set of abiotic and biotic stresses and subsequent effects on ectomycorrhizal (ECM) root tip colonization, seedling biomass, and leaf nitrogen content. The light regime had a more pronounced effect on ECM colonization than did juvenile damage. The interspecific competition resulted in higher ECM root tip abundance for Pinus, but this effect was insignificant in Fagus. Low light and interspecific competition resulted in lower seedling biomass compared to high light, and the effect of the latter was partially masked by high light. Leaf nitrogen responded differently in Fagus and Pinus when they grew in interspecific competition. Our results indicated that for both light-demanding (Pinus) and shade-tolerant (Fagus) species, the light environment was a major factor affecting seedling growth and ECM root tip abundance. The light conditions favorable for the growth of seedlings may to some extent compensate for the harmful effects of juvenile organ loss or damage and interspecific competition.

[Development of Arbuscular Mycorrhiza in Highly Responsive and Mycotrophic Host Plant-Black Medick (Medicago lupulina L.)].

The main phases of arbuscular mycorrhiza (AM) development were analyzed in black medick (Medicago lupulina) with Glomus intraradices. Methods of light and transmission electron microscopy were used to investigate AM. The first mycorrhization was identified on the seventh day after sowing. M. lupulina with AM-fungus Glomus intraradices formed Arum type of AM. Roots of black medick at fruiting stage (on the 88th day) were characterized by the development of forceful mycelium. The thickness of mycelium was comparable with the vascular system of root central cylinder. The development of vesicules into intraradical spores was shown. Micelium, arbuscules, and vesicules developed in close vicinity to the division zone of root tip. This might be evidence of an active symbiotic interaction between partners. All stages of fungal development and breeding, including intraradical spores (in inter-cellular matrix of root cortex), were identified in the roots of black medick, which indicated an active utilization of host plant nutrient substrates by the mycosymbiont. Plant cell cytoplasm extension was identified around young arbuscular branches but not for intracellular hyphae. The presence of active symbiosis was confirmed by increased accumulation of phosphorus in M. lupulina root tissues under conditions of G. intraradices inoculation and low phosphorus level in the soil. Thus, black medick cultivar-population can be characterized as an ecologically obligate mycotrophic plant under conditions of low level of available phosphorus in the soil. Specific features of AM development in intensively mycotrophic black medick, starting from the stage of the first true leaf until host plant fruiting, were evaluated. The obtained plant-microbe system is a perspective model object for further ultracytological and molecular genetic studies of the mechanisms controlling arbuscular mycorrhiza symbiotic efficiency, including selection and investigation of new symbiotic plant mutants.

Effect of retS gene on antibiotics production in Pseudomonas fluorescens FD6.

A hybrid sensor kinase termed RetS (regulator of exopolysaccharide and Type III secretion) controls expression of numerous genes in Pseudomonas aeruginosa. To investigate the function of RetS in P. fluorescens FD6, the retS gene was disrupted. Genetic inactivation of retS resulted in enhanced production of 2, 4-diacetylphloroglucinol, pyrrolnitrin, and pyoluteorin. The retS mutant also exhibited significant increase in phlA-lacZ, prnA-lacZ, and pltA-lacZ transcription levels, influencing expression levels of the small regulatory RNAs RsmX and RsmZ. In the gacSretS double mutant, all the phenotypic changes caused by the retS deletion were reversed to the level of gacS single mutant. Furthermore, the retS mutation drastically elevated biofilm formation and improved the colonization ability of strain FD6 on wheat rhizospheres. Based on these results, we proposed that RetS negatively controlled the production of antibiotics through the Gac/Rsm pathway in P. fluorescens FD6.