Elucidating ß-1,3-Glucanase and Peroxidase Physicochemical Properties of Wheat Cell Wall Defense Mechanism against Diuraphis noxia Infestation.

Wheat plants infested by Russian wheat aphids (RWA) induce a cascade of defense responses, including the hypersensitive responses (HR) and induction of pathogenesis-related (PR) proteins, such as ß-1,3-glucanase and peroxidase (POD). This study aims to characterize the physicochemical properties of cell wall-associated POD and ß-1,3-glucanase and determine their synergism on the cell wall modification during RWASA2-wheat interaction. The susceptible Tugela, moderately resistant Tugela-Dn1, and resistant Tugela-Dn5 cultivars were pregerminated and planted under greenhouse conditions, fertilized 14 days after planting, and irrigated every 3 days. The plants were infested with 20 parthenogenetic individuals of the same RWASA2 clone at the 3-leaf stage, and leaves were harvested at 1 to 14 days post-infestation. The Intercellular wash fluid (IWF) was extracted using vacuum filtration and stored at -20 °C. Leaf residues were crushed into powder and used for cell wall components. POD activity and characterization were determined using 5 mM guaiacol substrate and H2O2, monitoring change in absorbance at 470 nm. ß-1,3-glucanase activity, pH, and temperature optimum conditions were demonstrated by measuring the total reducing sugars in the hydrolysate with DNS reagent using ß-1,3-glucan and ß-1,3-1,4-glucan substrates, measuring the absorbance at 540 nm, and using glucose standard curve. The pH optimum was determined between pH 4 to 9, temperature optimum between 25 and 50 °C, and thermal stability between 30 °C and 70 °C. ß-1,3-glucanase substrate specificity was determined at 25 °C and 40 °C using curdlan and barley ß-1,3-1,4-glucan substrates. Additionally, the ß-1,3-glucanase mode of action was determined using laminaribiose to laminaripentaose. The oligosaccharide hydrolysis product patterns were qualitatively analyzed with thin-layer chromatography (TLC) and quantitatively analyzed with HPLC. The method presented in this study demonstrates a robust approach for infesting wheat with RWA, extracting peroxidase and ß-1,3-glucanase from the cell wall region and their comprehensive biochemical characterization.

FaERF2 activates two ß-1,3-glucanase genes to enhance strawberry resistance to Botrytis cinerea.

Ethylene response factor (ERF) is a class of plant-specific transcription factors that play an important role in plant growth, development, and stress response. However, the underlying mechanism of strawberry ERFs in pathogenic responses against Botrytis cinerea (B. cinerea) remains largely unclear. In this study, we isolated FaERF2, a nucleus-localized ERF transcription factor from Fragaria x ananassa. Transiently overexpressing FaERF2 in strawberry fruits significantly enhances their resistant ability to B. cinerea, while silencing FaERF2 in strawberry fruits enhances their susceptibility to B. cinerea. In addition, we found that FaERF2 could directly bind to the cis-acting element GCC box in the promoters of two ß-1,3-glucanase genes, FaBG-1 and FaBG-2, and activate their expression. Finally, both strawberry fruits transient expression followed by B. cinerea inoculation assays and recombinant protein incubation tests collectively substantiated the inhibitory effect of FaBG-1 and FaBG-2 on B. cinerea mycelium growth. These results revealed the molecular regulation mechanism of FaERF2 in response to B. cinerea and laid foundations for creating disease-resistance strawberry cultivar through genome editing approach.

Co-overexpression of chitinase and ß-1,3-glucanase significantly enhanced the resistance of Iranian wheat cultivars to Fusarium.

Fusarium head blight (FHB) is a devastating fungal disease affecting different cereals, particularly wheat, and poses a serious threat to global wheat production. Chitinases and ß-glucanases are two important proteins involved in lysing fungal cell walls by targeting essential macromolecular components, including chitin and ß-glucan micro fibrils. In our experiment, a transgenic wheat (Triticum aestivum) was generated by introducing chitinase and glucanase genes using Biolistic technique and Recombinant pBI121 plasmid (pBI-ChiGlu (-)). This plasmid contained chitinase and glucanase genes as well as nptII gene as a selectable marker. The expression of chitinase and glucanase was individually controlled by CaMV35S promoter and Nos terminator. Immature embryo explants from five Iranian cultivars (Arta, Moghan, Sisun, Gascogen and A-Line) were excised from seeds and cultured on callus induction medium to generate embryonic calluses. Embryogenic calluses with light cream color and brittle texture were selected and bombarded using gold nanoparticles coated with the recombinant pBI-ChiGlu plasmid. Bombarded calluses initially were transferred to selective callus induction medium, and later, they were transfferd to selective regeneration medium. The selective agent was kanamycin at a concentration of 25 mg/l in both media. Among five studied cultivars, A-Line showed the highest transformation percentage (4.8%), followed by the Sisun, Gascogen and Arta in descending order. PCR and Southern blot analysis confirmed the integration of genes into the genome of wheat cultivars. Furthermore, in an in-vitro assay, the growth of Fusarium graminearum was significantly inhibited by using 200 µg of leaf protein extract from transgenic plants. According to our results, the transgenic plants (T1) showed the resistance against Fusarium when were compared to the non-transgenic plants. All transgenic plants showed normal fertility and no abnormal response was observed in their growth and development.

Plant immunity suppression by an ß-1,3-glucanase of the maize anthracnose pathogen Colletotrichum graminicola.

BACKGROUND: Many phytopathogens secrete a large number of cell wall degrading enzymes (CWDEs) to decompose host cell walls in order to penetrate the host, obtain nutrients and accelerate colonization. There is a wide variety of CWDEs produced by plant pathogens, including glycoside hydrolases (GHs), which determine the virulence, pathogenicity, and host specificity of phytopathogens. The specific molecular mechanisms by which pathogens suppress host immunity remain obscure. RESULT: In this study, we found that CgEC124 encodes a glycosyl hydrolase with a signal peptide and a conserved Glyco_hydro_cc domain which belongs to glycoside hydrolase 128 family. The expression of CgEC124 was significantly induced in the early stage of Colletotrichum graminicola infection, especially at 12 hpi. Furthermore, CgEC124 positively regulated the pathogenicity, but it did not impact the vegetative growth of mycelia. Ecotopic transient expression of CgEC124 decreased the disease resistance and callose deposition in maize. Moreover, CgEC124 exhibited the ß-1,3-glucanase activity and suppresses glucan-induced ROS burst in maize leaves. CONCLUSIONS: Our results indicate that CgEC124 is required for full virulence of C. graminicola but not for vegetative growth. CgEC124 increases maize susceptibility by inhibiting host reactive oxygen species burst as well as callose deposition. Meanwhile, our data suggests that CgEC124 explores its ß-1,3-glucanase activity to prevent induction of host defenses.

Structural and functional analysis of SpGlu64A: a novel glycoside hydrolase family 64 laminaripentaose-producing ß-1,3-glucanase from Streptomyces pratensis.

Laminaripentaose (L5)-producing ß-1,3-glucanases can preferentially cleave the triple-helix curdlan into ß-1,3-glucooligosaccharides, especially L5. In this study, a newly identified member of the glycoside hydrolase family 64, ß-1,3-glucanase from Streptomyces pratensis (SpGlu64A), was functionally and structurally characterized. SpGlu64A shared highest identity (30%) with a ß-1,3-glucanase from Streptomyces matensis. The purified SpGlu64A showed maximal activity at pH 7.5 and 50 °C, and exhibited strict substrate specificity toward curdlan (83.1 U·mg-1). It efficiently hydrolyzed curdlan to produce L5 as the end product. The overall structure of SpGlu64A consisted of a barrel domain and a mixed (α/ß) domain, which formed an unusually wide groove with a crescent-like structure. In the two complex structures (SpGlu64A-L3 and SpGlu64A-L4), two oligosaccharide chains were captured and the triple-helical structure was relatively compatible with the wide groove, which suggested the possibility of binding to the triple-helical ß-1,3-glucan. A catalytic framework (ß6-ß9-ß10) and the steric hindrance formed by the side chains of residues Y161, N163, and H393 in the catalytic groove were predicted to complete the exotype-like cleavage manner. On the basis of the structure, a fusion protein with the CBM56 domain (SpGlu64A-CBM) and a mutant (Y161F; by site-directed mutation) were obtained, with 1.2- and 1.7-fold increases in specific activity, respectively. Moreover, the combined expression of SpGlu64A-CBM and -Y161F improved the enzyme activity by 2.63-fold. The study will not only be helpful in understanding the reaction mechanism of ß-1,3-glucanases but will also provide a basis for further enzyme engineering.

A conserved enzyme of smut fungi facilitates cell-to-cell extension in the plant bundle sheath.

Smut fungi comprise one of the largest groups of fungal plant pathogens causing disease in all cereal crops. They directly penetrate host tissues and establish a biotrophic interaction. To do so, smut fungi secrete a wide range of effector proteins, which suppress plant immunity and modulate cellular functions as well as development of the host, thereby determining the pathogen's lifestyle and virulence potential. The conserved effector Erc1 (enzyme required for cell-to-cell extension) contributes to virulence of the corn smut Ustilago maydis in maize leaves but not on the tassel. Erc1 binds to host cell wall components and displays 1,3-ß-glucanase activity, which is required to attenuate ß-glucan-induced defense responses. Here we show that Erc1 has a cell type-specific virulence function, being necessary for fungal cell-to-cell extension in the plant bundle sheath and this function is fully conserved in the Erc1 orthologue of the barley pathogen Ustilago hordei.

The lipopeptides fengycin and iturin are involved in the anticandidal activity of endophytic Bacillus sp. as determined by experimental and in silico analysis.

In this study, an endophytic Bacillus sp. strain (K7) was isolated from the medicinally important ornamental plant, Jasminum officinale. Biochemical analyses were conducted to evaluate the nature of the extracted product, which displayed strong anticandidal activity against Candida albicans (CA) SC5314, as evident from the results obtained in agar-cup diffusion tests, phase-contrast microscopy, scanning electron microscopy and minimum inhibitory concentration assays. After confirming the presence of the gene clusters encoding the lipopeptides iturins and fengycin in the genome of K7, their corresponding molecular ions were identified using MALDI-TOF-MS. 3D structures of the lipopeptides were downloaded from specific databases and molecular docking was performed against a vital CA enzyme, exo-1,3-beta-glucanase, involved in cell wall remodelling, adhesion to polymer materials and biofilm formation. The docking score of iturins was found to be -8·6 and -8·2 kcal mol-1 and for fengycin it was -9·4 kcal mol-1 , indicating a strong affinity of these cyclic lipopeptides towards exo-1,3-beta-glucanase. The combined in vitro and in silico anticandidal studies suggested that these secreted lipopeptides from Bacillus sp. may be used as potential therapeutics against opportunistic and complicated infections of CA.

Impact of Wheat Streak Mosaic Virus on Peroxisome Proliferation, Redox Reactions, and Resistance Responses in Wheat.

Although peroxisomes play an essential role in viral pathogenesis, and viruses are known to change peroxisome morphology, the role of genotype in the peroxisomal response to viruses remains poorly understood. Here, we analyzed the impact of wheat streak mosaic virus (WSMV) on the peroxisome proliferation in the context of pathogen response, redox homeostasis, and yield in two wheat cultivars, Patras and Pamir, in the field trials. We observed greater virus content and yield losses in Pamir than in Patras. Leaf chlorophyll and protein content measured at the beginning of flowering were also more sensitive to WSMV infection in Pamir. Patras responded to the WSMV infection by transcriptional up-regulation of the peroxisome fission genes PEROXIN 11C (PEX11C), DYNAMIN RELATED PROTEIN 5B (DRP5B), and FISSION1A (FIS1A), greater peroxisome abundance, and activation of pathogenesis-related proteins chitinase, and ß-1,3-glucanase. Oppositely, in Pamir, WMSV infection suppressed transcription of peroxisome biogenesis genes and activity of chitinase and ß-1,3-glucanase, and did not affect peroxisome abundance. Activity of ROS scavenging enzymes was higher in Patras than in Pamir. Thus, the impact of WMSV on peroxisome proliferation is genotype-specific and peroxisome abundance can be used as a proxy for the magnitude of plant immune response.

Evaluating ß-1,3-glucan synthesis inhibition using emulsion formation as an indicator.

INTRODUCTION: The cell wall ß-1,3-glucan of fungal pathogen Candida albicans is an attractive antifungal target. ß-1,3-Glucan is the skeletal structure in the cell wall and the major scaffold for cell wall proteins. In previous studies using Saccharomyces cerevisiae, strong emulsification was detected by mixing cell wall proteins with oil. To date, there have been no reports of applying an emulsification phenomenon to assessing ß-1,3-glucan synthesis inhibition. OBJECTIVE: The aim of this study was to clarify that emulsification is useful as an indicator for evaluating ß-1,3-glucan synthesis inhibition in C. albicans. METHODS: At first, whether cell wall proteins released from cells by ß-1,3-glucanase treatment worked as an effective emulsifier in C. albicans was examined. Next, whether emulsification occurred even in the culture supernatant brought about by treating with bioactive compounds, including ß-1,3-glucan synthesis inhibitors, under osmotic protection was investigated. In addition, the release of cell wall proteins into the culture medium by treating with those compounds was examined. Finally, a simpler evaluation method using emulsion formation was examined for application to screening of inhibitors. RESULTS: Emulsification occurred by cell wall proteins obtained by treating with ß-1,3-glucanase in C. albicans. In addition, cell wall proteins were released into the culture medium by treating with ß-1,3-glucan synthesis inhibitors, resulting in emulsification. However, such phenomena were not observed in the case of other bioactive compounds. Furthermore, emulsification could be detected in the culture broth obtained by static culture on a small scale. CONCLUSIONS: The obtained results strongly implied that emulsification results from decreased ß-1,3-glucan levels in the cell wall. As emulsification can be simply evaluated by mixing the culture broth with oil, in the future application to the initial assessment and screening of ß-1,3-glucan synthesis inhibitors is expected.

Purification of a killer toxin from Aureobasidium pullulans for the biocontrol of phytopathogens.

The objectives of the present study were to purify and assess the killer toxin effect produced by Aureobasidium pullulans under casual agents of green mold (Penicillum digitatum) and sour rot (Geotrichum citri-aurantii). Initially, different methods of protein precipitation were tested. The proteolytic activity and the presence of proteins acting on cell wall receptors, ß-1,3-glucanase and chitinase were determined, and toxin purification was conducted by Sephadex G-75 gel exclusion chromatography and cellulose chromatography (medium fibers). Subsequently, purification was confirmed by polyacrylamide gel electrophoresis, and the detection of killer activity was performed in solid YEPD-methylene blue buffered with citrate-phosphate (0.1 M, pH 4.6). Toxin identification was performed by liquid chromatography-mass spectrometry. The results showed that the best protein precipitation method was 2:1 ethanol (vol/vol ethanol/supernatant). It was possible to observe the presence of enzymes with proteolytic activity, including ß-1,3-glucanase and chitinase. During the purification process, it was verified that the killer toxin produced by the yeast has a low-molecular-weight protein belonging to the ubiquitin family, which presents killer activity against P. digitatum and G. citri-aurantii.

Structural insights into ß-1,3-glucan cleavage by a glycoside hydrolase family.

The fundamental and assorted roles of ß-1,3-glucans in nature are underpinned on diverse chemistry and molecular structures, demanding sophisticated and intricate enzymatic systems for their processing. In this work, the selectivity and modes of action of a glycoside hydrolase family active on ß-1,3-glucans were systematically investigated combining sequence similarity network, phylogeny, X-ray crystallography, enzyme kinetics, mutagenesis and molecular dynamics. This family exhibits a minimalist and versatile (α/ß)-barrel scaffold, which can harbor distinguishing exo or endo modes of action, including an ancillary-binding site for the anchoring of triple-helical ß-1,3-glucans. The substrate binding occurs via a hydrophobic knuckle complementary to the canonical curved conformation of ß-1,3-glucans or through a substrate conformational change imposed by the active-site topology of some fungal enzymes. Together, these findings expand our understanding of the enzymatic arsenal of bacteria and fungi for the breakdown and modification of ß-1,3-glucans, which can be exploited for biotechnological applications.

Effect of heat shock and potassium sorbate treatments on gray mold and postharvest quality of 'XuXiang' kiwifruit.

We determined whether heat and chemical treatments could reduce the decay of kiwifruit caused by Botrytis cinerea during postharvest storage. Kiwifruits were treated with 5 g/L (w/v) potassium sorbate (PS), with a 48 °C hot water treatment (HT), and with a combined treatment (HT + PS). Mycelial growth of B. cinerea and the postharvest quality of 'XuXiang' kiwifruits were evaluated. HT + PS significantly inhibited mycelial growth, germ tube growth, and spore germination of B. cinerea. This treatment also reduced the incidence of gray mold in kiwifruit postharvest, and enhanced activities of defense-related enzymes in kiwifruit tissues. Compared with the control, all treatments resulted in lower malondialdehyde (MDA) contents and higher total phenolic contents in kiwifruits. HT + PS also increased the activities of chitinase and ß-1,3-glucanase and the transcript levels of their encoding genes. HT + PS can improve kiwifruit quality and reduce decay during postharvest storage.

Discovery of hyperstable carbohydrate-active enzymes through metagenomics of extreme environments.

The enzymes from hyperthermophilic microorganisms populating volcanic sites represent interesting cases of protein adaptation and biotransformations under conditions where conventional enzymes quickly denature. The difficulties in cultivating extremophiles severely limit access to this class of biocatalysts. To circumvent this problem, we embarked on the exploration of the biodiversity of the solfatara Pisciarelli, Agnano (Naples, Italy), to discover hyperthermophilic carbohydrate-active enzymes (CAZymes) and to characterize the entire set of such enzymes in this environment (CAZome). Here, we report the results of the metagenomic analysis of two mud/water pools that greatly differ in both temperature and pH (T = 85 °C and pH 5.5; T = 92 °C and pH 1.5, for Pool1 and Pool2, respectively). DNA deep sequencing and following in silico analysis led to 14 934 and 17 652 complete ORFs in Pool1 and Pool2, respectively. They exclusively belonged to archaeal cells and viruses with great genera variance within the phylum Crenarchaeota, which reflected the difference in temperature and pH of the two Pools. Surprisingly, 30% and 62% of all of the reads obtained from Pool1 and 2, respectively, had no match in nucleotide databanks. Genes associated with carbohydrate metabolism were 15% and 16% of the total in the two Pools, with 278 and 308 putative CAZymes in Pool1 and 2, corresponding to ~ 2.0% of all ORFs. Biochemical characterization of two CAZymes of a previously unknown archaeon revealed a novel subfamily GH5_19 ß-mannanase/ß-1,3-glucanase whose hemicellulose specificity correlates with the vegetation surrounding the sampling site, and a novel NAD+ -dependent GH109 with a previously unreported ß-N-acetylglucosaminide/ß-glucoside specificity. DATABASES: The sequencing reads are available in the NCBI Sequence Read Archive (SRA) database under the accession numbers SRR7545549 (Pool1) and SRR7545550 (Pool2). The sequences of GH5_Pool2 and GH109_Pool2 are available in GenBank database under the accession numbers MK869723 and MK86972, respectively. The environmental data relative to Pool1 and Pool2 (NCBI BioProject PRJNA481947) are available in the Biosamples database under the accession numbers SAMN09692669 (Pool1) and SAMN09692670 (Pool2).

Bacillomycin D inhibits growth of Rhizopus stolonifer and induces defense-related mechanism in cherry tomato.

The inhibitory effect of Bacillomycin D, a cyclic lipopeptide, on Rhizopus stolonifer colonization of cherry tomato was studied, and its possible mechanism of action was explored. Bacillomycin D showed a direct inhibitory effect on R. stolonifer spore germination and mycelial growth in vitro. It conferred both a direct inhibitory effect on R. stolonifer growth in cherry tomato in vivo and induced host resistance in cherry tomato. Moreover, Bacillomycin D treatment significantly increased the activities of plant defense-related enzymes, including chitinase (CHI), ß-1,3-glucanase (GLU), phenylalanine ammonia-lyase (PAL), and peroxidase (POD). Real-time PCR (RT-PCR) showed that defense-related genes involved in the salicylic acid defense signaling pathway and genes encoding pathogenesis-related proteins were up-regulated in Bacillomycin D treatment. Furthermore, Bacillomycin D-C16 resulted in direct inhibition and a remarkable induced resistance to R. stolonifer which was higher than as induced by Bacillomycin D-C14. Together, the data indicated that Bacillomycin D can control the growth of R. stolonifer through both the direct inhibition of the fungus and the activation of defense-related genes and enzymes in cherry tomato.

Conformation and cooperative order-disorder transition in aqueous solutions of ß-1,3-d-glucan with different degree of branching varied by the Smith degradation.

ß-1,3-d-glucan with different degrees of branching were obtained by selectively and gradually removing side chains from schizophyllan, a water-soluble triple helical polysaccharide, using the Smith degradation. Size exclusion chromatography combined with a multi-angle light scattering detection was performed in aqueous 0.1 M NaCl. The degree of branching decreased after the Smith degradation, while the molar mass distributions were almost unchanged. The molecular conformation of the Smith-degraded ß-1,3-d-glucan was analyzed on the basis of the molar mass dependency of the radius gyration, and found to be comparable to the original triple helix of schizophyllan. Differential scanning calorimetry in deuterium oxide-hexadeuterodimethylsulfoxide mixtures was performed to investigate the effects of the degree of branching on the cooperative order-disorder transition. Removal of side chains affects both the transition temperature and transition enthalpy. The ordered structure is formed by the residual side chains in the triplex unit, so that the linear cooperative system of the triplex is maintained after the Smith degradation.

Purification and characterization of a novel ß-1,3-glucanase from Arca inflata and its immune-enhancing effects.

A novel ß-1,3-glucanase from Arca inflata was purified using chromatography methods. It was determined as a glycoprotein comprising 23.65% carbohydrate content with O-linked glycan and showed specific activity of 90.01 ±â€¯1.2 U/mg against laminarin. The optimal pH and temperature for the activity of the glucanase were 6.0 and 40 °C, respectively. The affinity parameter of the glucanase using laminarin was determined as Kd = 13.09 µM. The activity of the glucanase was 27 ±â€¯2.6% enhanced by 2-mM Mn2+ ions and inhibited by 40-50% using 2-mM Zn2+, Cu2+, or Ba2+ ions. The glucanase showed an endo-type cleavage mode and hydrolyzed laminarin into glucoses, disaccharides, trioligosaccharides, and tetraoligosaccharides. Otherwise, the glucanase exhibited immune-enhancing effects via significantly increasing the phagocytic activity of macrophages and inducing the release of nitric oxide, tumor necrosis factor α, and interleukin-6 in RAW264.7 cells. It might be used as a bifunctional additive for the food industry.

Curdlan conformation change during its hydrolysis by multi-domain ß-1,3-glucanases.

Enzymatic curdlan hydrolysis is gaining more attention for the value of oligo-ß-glucans in many aspects. Currently, the triple-helical conformation of curdlan fiber was imposed to the structure of ß-1,3-glucanase as its substrate without experimental evidence. Here, solution conformation of differently treated curdlan and each hydrolysis rate by a variety of ß-1,3-glucanases were systematically examined. Results showed that different enzymes exhibited preferences over the trajectories of pH change that curdlan solution went through, and all enzymes hydrolyzed heat treated curdlan solution at their maximum rates where most of the higher ordered helices were diminished. Combined with molecular docking studies, a multi-step hydrolysis process was proposed. Recognition of triple-helical curdlan by their ancillary region of ß-1,3-glucanase occurred before its unwinding into single- and double-helical forms, and the later ones fitted better to the catalytic cavity of the enzyme where the polysaccharides chain eventually got hydrolyzed into oligo-ß-glucans.

A novel chitosan formulation treatment induces disease resistance of harvested litchi fruit to Peronophythora litchii in association with ROS metabolism.

This study aimed to investigate the effects of a novel chitosan formulation (Kadozan) treatment on disease development, response of disease resistance, metabolism of reactive oxygen species (ROS) in Peronophthora litchii-inoculated "Wuye" litchis. Compared with P. litchii-inoculated litchis, Kadozan-treated P. litchii-inoculated litchis exhibited lower fruit disease index, higher lignin content, higher activities of disease resistance-related enzymes (CHI, GLU and PAL), lower O2- generating rate and malondialdehyde content, higher activities of ROS scavenging enzymes (SOD, CAT and APX), higher contents of ascorbic acid and glutathione, and higher levels of reducing power and DPPH radical scavenging activity. These results suggest that Kadozan can be used to inhibit the growth of P. litchii in harvested litchis owning to the enhancement of disease resistance and ROS scavenging capacity, and decreases in O2- accumulation and membrane lipid peroxidation. Kadozan treatment can be used as a facile and novel method for suppressing postharvest pathogenic disease of litchis.

Callose-Regulated Symplastic Communication Coordinates Symbiotic Root Nodule Development.

The formation of nitrogen-fixing nodules in legumes involves the initiation of synchronized programs in the root epidermis and cortex to allow rhizobial infection and nodule development. In this study, we provide evidence that symplastic communication, regulated by callose turnover at plasmodesmata (PD), is important for coordinating nodule development and infection in Medicago truncatula. Here, we show that rhizobia promote a reduction in callose levels in inner tissues where nodules initiate. This downregulation coincides with the localized expression of M. truncatula ß-1,3-glucanase 2 (MtBG2), encoding a novel PD-associated callose-degrading enzyme. Spatiotemporal analyses revealed that MtBG2 expression expands from dividing nodule initials to rhizobia-colonized cortical and epidermal tissues. As shown by the transport of fluorescent molecules in vivo, symplastic-connected domains are created in rhizobia-colonized tissues and enhanced in roots constitutively expressing MtBG2. MtBG2-overexpressing roots additionally displayed reduced levels of PD-associated callose. Together, these findings suggest an active role for MtBG2 in callose degradation and in the formation of symplastic domains during sequential nodule developmental stages. Interfering with symplastic connectivity led to drastic nodulation phenotypes. Roots ectopically expressing ß-1,3-glucanases (including MtBG2) exhibited increased nodule number, and those expressing MtBG2 RNAi constructs or a hyperactive callose synthase (under symbiotic promoters) showed defective nodulation phenotypes. Obstructing symplastic connectivity appears to block a signaling pathway required for the expression of NODULE INCEPTION (NIN) and its target NUCLEAR FACTOR-YA1 (NF-YA1) in the cortex. We conclude that symplastic intercellular communication is proactively enhanced by rhizobia, and this is necessary for appropriate coordination of bacterial infection and nodule development.

Exposure to heavy metal stress triggers changes in plasmodesmatal permeability via deposition and breakdown of callose.

Both plants and animals must contend with changes in their environment. The ability to respond appropriately to these changes often underlies the ability of the individual to survive. In plants, an early response to environmental stress is an alteration in plasmodesmatal permeability with accompanying changes in cell to cell signaling. However, the ways in which plasmodesmata are modified, the molecular players involved in this regulation, and the biological significance of these responses are not well understood. Here, we examine the effects of nutrient scarcity and excess on plasmodesmata-mediated transport in the Arabidopsis thaliana root and identify two CALLOSE SYNTHASES and two ß-1,3-GLUCANASES as key regulators of these processes. Our results suggest that modification of plasmodesmata-mediated signaling underlies the ability of the plant to maintain root growth and properly partition nutrients when grown under conditions of excess nutrients.