The plasmodesmata-associated β-1,3-glucanase gene GhPdBG regulates fiber development in cotton

Trichomes are specialized structures that originate from epidermal cells of organs in higher plants.The cotton fiber is a unique single-celled trichome that elongates from the seed coat epidermis.Cotton(Gossypium hirsutum)fibers and trichomes are models for cell differentiation.In an attempt to elucidate the intercellular factors that regulate fiber and trichome cell development,we identified a plasmodesmal β-1,3-glucanase gene(designated GhPdBG)controlling the opening and closing of plasmodesmata in cotton fibers.Structural and evolutionary analysis showed haplotypic variation in the promoter region of the GhPdBG gene among 352 cotton accessions,but high conservation in the coding region.GhPdBG was expressed predominantly in cotton fibers and localized to plasmodesmata(PD).Expression patterns of PdBG that corresponded to PD permeability were apparent during fiber development in G.hirsutum and G.barbadense.The PdBG-mediated opening-closure of PD appears to be involved in fiber development and may account for the contrasting fiber traits of these two species.Ectopic expression of GhPdBG revealed that it functions in regulating fiber and trichome length and/or density by modulating plasmodesmatal permeability.This finding suggests that plasmodesmal targeting of GhPdBG,as a switch of intercellular channels,regulates single-celled fiber and trichome development in cotton.

Cloning and Bioinformatics Analysis of Rosa rugosa β-1,3-Glucanase Gene (RrGlu)

In order to reveal which role the callose played in R. rugosa pollination incompatibility, the full-length cDNA sequence of β-1,3-glucanase gene was cloned for the first time from the stylus of Rosa rugosa “Tanghong” with RT-PCR and RACE methods and named as RrGlu. The full-length cDNA is 1380 bp with an open reading frame of 1041 bp, encoding 346 amino acids. The derived protein has a molecular weight of 37.85 kD, a calculated pI of 9.12, a pfam00332 conserved domain at position 36 - 345, and belongs to glycosyl hydrolase family 17. The derived protein is a hydrophilic protein secreted into the vacuole. There is a signal peptide cleavage site at position 34 - 35, a transmembrane domain at position 13 - 32, six Ser phosphorylation sites, three Thr phosphorylation sites, three Tyr phosphorylation sites, one N-glycosylation site, and five O-glycosylation sites. There are 31.50% α-helixes, 30.92% random coil, 25.14% extended peptide chain, and 12.43% β-corner structure. This protein and the Glu protein from eight other species, including Prunus persica, share a sequence homology of greater than 72%;all of the proteins contain a pfam00332 conserved domain and a β-1,3-glucanase active center sequence (LIVM)-X-(LIVMFYW)3-(STAG)-E-(ST)-G-W-P-(ST)-X-G. Furthermore, their phylogenetic relationships are consistent with their traditional classifications. These results were meaningful to reveal the molecular mechanism of R. rugosa pollination incompatibility and improve the theory and techniques of breeding ornamental R. rugosa.

Partitioning and purification of extracellular β-1,3-1,4-glucanase in aqueous two-phase systems

The partition behaviors of β-1,3-1,4-glucanase, α-amylase and neutral proteases from clarified and whole fermentation broths of Bacillus subtilis ZJF-1A5 were investigated. An aqueous two-phase system （polyethylene glycol （PEG）/MgSO4） was examined with regard to the effects of PEG molecular weight （MW） and concentration, MgSO4 concentration, pH and NaC1 concentration on enzyme partition and extraction. The MW and concentration of PEG were found to have significant effects on enzyme partition and extraction with low MW PEG showing the greatest benefit in the partition and extraction of β-glucanase with the PEG/MgSO4 system. MgSO4 concentration influenced the partition and extraction of β-glucanase significantly, pH had little effect on β-glucanase or proteases partition but affected a-amylase partition when pH was over 7.0. The addition of NaCl had little effect on the partition behavior of β-glucanase but had very significant effects on the partitioning of α-amylase and on the neutral proteases. The partition behaviors of β-glucanase, α-amylase and proteases in whole broth were also investigated and results were similar to those obtained with clarified fermentation broth. A two-step process for purifying β-glucanase was developed, which achieved β-glucanase recovery of 65.3% and specific activity of 14027 U/mg, 6.6 times improvement over the whole broth.

Changes in Protein Content and Chitinase and β-1,3- glucanase Activities of Rice with Blast Resistance Induced by Ag-antibiotic 702

[Objective] The paper was to explore the effect of rice blast resistance induced by Ag-antibiotics 702 on protein content and chitlnase and β-1,3-glucanase activities in rice. [ Method] At the fourth leaf stage of Luliangyou 996, 15 μg/mL Ag-antibiotic 702 was sprayed, while Validamycin and distilled wa- ter were sprayed as positive control and negative control, respectively. All treatments were inoculated with spore fluid of Magnaporthe grisea at 48 h post spraying, and the rice inoculated with only distilled water was used as blank control. The enzymes activities （endochitinase, exochitinase and β-1,3-glucanase） and total pro- tein content in rice leaves were determined every 24 h within 168 h post spraying. [ Result] Compared with the blank control, the rice inoculated with spore fluid of M. grisea could significantly increase the total protein content and the activities of β-1,3-glucanase and chitinase. The induction effect of Ag-antibiotics 702 exceeded that of Validamycin treatment. And the changes in activities of β-1,3-glucanase and chitinase had obvious synchronicity. [ Conclusion] Ag-antibiotic 702 can significantly improve the total protein content and the activities of β-1,3-glucanase and chitinase, thus enhancing the resistance to rice blast.

Increase of β -1, 3-Glucanase and Chitinase Activities in Cotton Callus Cells Treated by Salicylic Acid and Toxin of Verticillium dahliae

The different resistance of cotton (Gossypium hirsutum L.) cultivars to crude toxin of Verticillium dah/iae(VD) was correlated with the activities of chitinase and β-1, 3-glucanase in callus cells. The activities of chitinase and β-1, 3-glucanase in the callus cells treated with the VD-toxin were increased to the higher level at earlier time point in resistant cultivars than these in the susceptible cultivars. Exogenous salicylic acid (SA) induced the accumulation of chitinase and β -1,3-glucanase, which resulted in the resistance of callus cells to the VD. toxin. Western blot using a polyclonal antibody against β -1,3-glucanase identified 28 kD protein that was induced by VD-toxin, SA, or VD-toxin plus SA.

Induction of Volatile Organic Compounds in Leaves of Lycopersicon Esculentum by Nd^(3+)

The effects of Nd^3＋ on the quality and quantity of volatile organic compounds （VOCs） in the leaves of Lycopersicon esculentum were studied. The results demonstrate that Nd^3＋ can increase the total amount of VOC by 75% after treatment for 120 h, as compared with the control. Phyto-oxylipins, terpenoids and aromatic compounds were increased by 73%, 38% and 21%, respectively. （E）-2-hexenal, the most abundant constituent is increased by 74%, β- phellandrene and α-caryophyllene in terpenoids,

Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination

The bglS gene encoding endo-1,3-1,4-β-glucanase from Bacillus subtil＆ was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone a-factor （MFals）, and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction （PCR）-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae （SC-βG） was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A （PrA） activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-1,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-1,3-1,4-β-glucanase expression level with the maximum of 69.3 U/（h·ml） after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-1,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.

Antifungal Potential of Beauveria bassiana on Solanum lycopersicum L. Infected with Fusarium oxysporum f. sp. lycopersici

The objective of this work was to evaluate the effect of Beauveria bassiana(Bb 1205)on controlling Fusarium oxysporum f.sp.lycopersici(Fol 17108)in tomato plants in greenhouse conditions.Inoculation of Bb 1205 was the most promising among the agronomic variables and expression of the activity of the enzymesβ-1,3-glucanases and chitinases.Inoculation of Bb 1205 occurred at a concentration of 1×108 conidia·mL−1,which was administered onto the leaves,directly into the soil and via injection.Infection with Fol 17108 occurred with 1×106 spores·mL−1,which were added directly to the soil.Spectrophotometry was used for measuring agronomic parameters,namely activity of chitinases andβ-1,3-glucanases in foliage and roots.When Bb 1205 was added to the soil,the chlorophyll index and aerial part length showed significant differences.In addition,it was determined that root length,fresh weight of foliage,flower,and fruit count increased 82 days after inoculation(dai).Chitinase activity induced by Bb 1205 in leaves and roots of tomato plants infected with Fol 17108 was observed when injected into the stem at 32 dai(41.8 and 11.6-fold,respectively).Inoculation on the foliage showed a 10-fold increase ofβ-1,3-glucanases in the roots after 82 dpi.As for leaves,a 3.8-fold increase was found when the stem was inoculated.In the different in vivo applications,Bb 1205 activated its defenses by expressing the chitinase enzymes andβ-1,3-glucanase,thus reducing the damage caused by Fol 17108,demonstrating increase plant growth thereafter.

Stem Blight Control of Schizonepeta tenuifolia Caused by Phytophthora nicotianae Using Trichoderma spp.

Objective To control stem blight disease of Schizonepeta tenuifolia caused by Phytophthora nicotianae. Methods The antagonist effect of 13 Trichoderma strains (including T. viride and T. harzianum) was evaluated upon mycelia growth of P. nicotianae. Trichoderma strains with high antagonistic activities against the pathogen were used to control stem blight of S. tenuifolia in the field. Results Of 13 Trichoderma strains tested, T. viride strain M3 showed maximum mycelia growth inhibition (83.2%) to the pathogen, followed by T. viride strain Tv04-2 (78.2%) and then T. harzianum strain ThB (65.0%), in vitro. Fungal cell wall degrading enzymes, protease, and β-1,3-glucanase were analyzed qualitatively and quantitatively in further study. T. viride strains M3, Tv04-2, and T. harzianum strain ThB efficiently against P. nicotianae were used to control stem blight of S. tenuifolia in the field, and T. viride strain M3 showed the best biocontrol potential. Conclusion Trichoderma spp. can be used as alternatives of pesticides to control stem blight, one of the serious soilborne diseases of S. tenuifolia caused by P. nicotianae. However, though T. viride strains Tv04-2 and T. harzianum strain ThB are also highly against P. nicotianae in vitro, the controlling efficacy of them on stem blight disease is not as excellent as T. viride strains M3 in the field.