Characterization and improvement of curdlan produced by a high-yield mutant of Agrobacterium sp. ATCC 31749 based on whole-genome analysis.

Curdlan is a bacterial, water-insoluble, linear homopolysaccharide that has been widely used in the food industry. In this study, genome information of strain CGMCC 11546, a UV-induced high-yield mutant of the model curdlan-producing strain Agrobacterium sp. ATCC 31749, was used to investigate the molecular mechanism of curdlan biosynthesis. The maximum curdlan yield of 47.97 ± 0.57 g/L was obtained from strain CGMCC 11546 by using optimal media containing 60 g/L sucrose, 6 g/L yeast, 2 g/L KH2PO4, 0.4 g/L MgSO4·7H2O, 2 g/L CaCO3, 0.1 g/L FeSO4·7H2O, 0.04 g/L MnSO4, and 0.02 g/L ZnCl2 at 30 °C and 280 rpm after 96 h of fermentation. The gel strength of curdlan was improved by 41 % by knocking out the ß-1,3-glucanase genes exoK and exsH of strain CGMCC 11546. Furthermore, the application of curdlan from the ΔexoK-exsH strain in noodles significantly improved the eating quality of both raw and cooked noodles.

Improved extraction of resveratrol and antioxidants from grape peel using heat and enzymatic treatments.

BACKGROUND: Resveratrol, an extensively recognized phytochemical that belongs to the stilbene family, is abundant in grape peel which is discarded as a by-product during grape juice processing. RESULTS: In this study, we established that pre-heating grape peel above 75 °C significantly improved the extractability of resveratrol and its glucoside piceid. In particular, thermal heating of grape peel at 95 °C for 10 min, followed by treatment with a mixture of exo-1,3-ß-glucanase and pectinases at 50 °C for 60 min, dramatically increased the conversion of piceid into resveratrol and the overall extractability of this phytochemical by 50%. Furthermore, thermal pre-treatment promoted a substantial increase in the total phenol, flavonoid, and anthocyanin concentrations in the grape peel extract. Ultimately, resveratrol-enriched grape peel extract significantly augmented the antioxidant response in vitro, possibly by attenuating the accumulation of intracellular reactive oxygen species via the Nrf2 signaling pathway. CONCLUSION: The method developed in this study for preparing grape peel extract introduces a potential low-cost green processing for the industrial fortification of food products with resveratrol and other health-beneficial antioxidants. © 2019 Society of Chemical Industry.

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.

The C-terminal domains of two homologous Oleaceae ß-1,3-glucanases recognise carbohydrates differently: Laminarin binding by NMR.

Ole e 9 and Fra e 9 are two allergenic ß-1,3-glucanases from olive and ash tree pollens, respectively. Both proteins present a modular structure with a catalytic N-terminal domain and a carbohydrate-binding module (CBM) at the C-terminus. Despite their significant sequence resemblance, they differ in some functional properties, such as their catalytic activity and the carbohydrate-binding ability. Here, we have studied the different capability of the recombinant C-terminal domain of both allergens to bind laminarin by NMR titrations, binding assays and ultracentrifugation. We show that rCtD-Ole e 9 has a higher affinity for laminarin than rCtD-Fra e 9. The complexes have different exchange regimes on the NMR time scale in agreement with the different affinity for laminarin observed in the biochemical experiments. Utilising NMR chemical shift perturbation data, we show that only one side of the protein surface is affected by the interaction and that the binding site is located in the inter-helical region between α1 and α2, which is buttressed by aromatic side chains. The binding surface is larger in rCtD-Ole e 9 which may account for its higher affinity for laminarin relative to rCtD-Fra e 9.

An Enzymatically Active ß-1,3-Glucanase from Ash Pollen with Allergenic Properties: A Particular Member in the Oleaceae Family.

Endo-ß-1,3-glucanases are widespread enzymes with glycosyl hydrolitic activity involved in carbohydrate remodelling during the germination and pollen tube growth. Although members of this protein family with allergenic activity have been reported, their effective contribution to allergy is little known. In this work, we identified Fra e 9 as a novel allergenic ß-1,3-glucanase from ash pollen. We produced the catalytic and carbohydrate-binding domains as two independent recombinant proteins and characterized them from structural, biochemical and immunological point of view in comparison to their counterparts from olive pollen. We showed that despite having significant differences in biochemical activity Fra e 9 and Ole e 9 display similar IgE-binding capacity, suggesting that ß-1,3-glucanases represent an heterogeneous family that could display intrinsic allergenic capacity. Specific cDNA encoding Fra e 9 was cloned and sequenced. The full-length cDNA encoded a polypeptide chain of 461 amino acids containing a signal peptide of 29 residues, leading to a mature protein of 47760.2 Da and a pI of 8.66. An N-terminal catalytic domain and a C-terminal carbohydrate-binding module are the components of this enzyme. Despite the phylogenetic proximity to the olive pollen ß-1,3-glucanase, Ole e 9, there is only a 39% identity between both sequences. The N- and C-terminal domains have been produced as independent recombinant proteins in Escherichia coli and Pichia pastoris, respectively. Although a low or null enzymatic activity has been associated to long ß-1,3-glucanases, the recombinant N-terminal domain has 200-fold higher hydrolytic activity on laminarin than reported for Ole e 9. The C-terminal domain of Fra e 9, a cysteine-rich compact structure, is able to bind laminarin. Both molecules retain comparable IgE-binding capacity when assayed with allergic sera. In summary, the structural and functional comparison between these two closely phylogenetic related enzymes provides novel insights into the complexity of ß-1,3-glucanases, representing a heterogeneous protein family with intrinsic allergenic capacity.

Relationship between male sterility and ß-1,3-glucanase activity and callose deposition-related gene expression in wheat (Triticum aestivum L.).

In previous studies, we first isolated one different protein ß-1,3-glucanase using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry from normal wheat (Triticum aestivum L.) and chemical hybridization agent-induced male sterility (CIMS) wheat. In this experiment, ß-1,3-glucanase activity and the expression of a callose deposition-related gene, UDP-glucose phosphorylase (UGPase), were determinate in normal, CIMS, and genetic male sterility (GS) wheat. ß-1,3-glucanase activity was significantly different between the fertile and sterile lines during callose synthesis and degradation, but there was no difference between CIMS and GS wheat. The UGPase gene of callose deposition was highly expressed in the meiophase and sharply decreased in the tetrad stage. However, the expression of the UGPase gene was significantly different between the fertile and sterile lines. These data indicated that ß-1,3-glucanase activity and the expression of the UGPase gene play important roles in the male sterility of wheat. Consequently, pollen mother cells (PMCs) might degenerate at the early meiosis stage, and differences in UGPase gene expression and ß-1,3-glucanase activity might eventually result in complete pollen collapse. In addition, the critical period of anther abortion might be the meiosis stage to the tetrad stage rather than what we previously thought, the mononuclear period.

Cloning and functional characterization of ß-1, 3-glucanase gene from Podophyllum hexandrum - a high altitude Himalayan plant.

Podophyllum hexandrum is a high-altitude medicinal plant exploited for its etoposides which are potential anticancer compounds. ß-1, 3-glucanase cDNA was cloned from the germinating seeds of Podophyllum (Ph-glucanase). Glucanases belong to pathogenesis related glycohydralase family of proteins, which also play an important role in endosperm weakening and testa rupture during seed germination. Analysis of cloned nucleotide sequence revealed Ph-glucanase with an open reading frame of 852bp encoding a protein of 283 amino acids with a molecular mass of 31kDa and pI of 4.39. In-silico structure prediction of Ph-glucanase showed homology with that of Hevea brasiliensis (3em5B). Structural stability and enhanced catalytic efficiency in harsh climatic conditions possibly due to the presence of glycosyl hydrolase motif (LGIVISESGWPSAG) and a connecting loop towards inner side and well exposed carbohydrate metabolism domain-COG5309, can readily hydrolyse cell wall sugar moieties. Seeds from the transgenic Arabidopsis plants over-expressing Ph-glucanase showed better germination performance against a wide range of temperatures and abscisic acid (ABA) stress. This can be attributed to the accumulation of Ph-glucanase at both transcript and protein levels during the seed germination in transgenic Arabidopsis. Results confirm that the cloned novel seed specific glucanase from a cold desert plant Podophyllum could be used for the manipulation of different plant species seeds against various harsh conditions.

The Arabidopsis CALLOSE DEFECTIVE MICROSPORE1 gene is required for male fertility through regulating callose metabolism during microsporogenesis.

During angiosperm microsporogenesis, callose serves as a temporary wall to separate microsporocytes and newly formed microspores in the tetrad. Abnormal callose deposition and dissolution can lead to degeneration of developing microspores. However, genes and their regulation in callose metabolism during microsporogenesis still remain largely unclear. Here, we demonstrated that the Arabidopsis (Arabidopsis thaliana) CALLOSE DEFECTIVE MICROSPORE1 (CDM1) gene, encoding a tandem CCCH-type zinc finger protein, plays an important role in regulation of callose metabolism in male meiocytes and in integrity of newly formed microspores. First, quantitative reverse transcription PCR and in situ hybridization analyses showed that the CDM1 gene was highly expressed in meiocytes and the tapetum from anther stages 4 to 7. In addition, a transfer DNA insertional cdm1 mutant was completely male sterile. Moreover, light microscopy of anther sections revealed that microspores in the mutant anther were initiated, and then degenerated soon afterward with callose deposition defects, eventually leading to male sterility. Furthermore, transmission electron microscopy demonstrated that pollen exine formation was severely affected in the cdm1 mutant. Finally, we found that the cdm1 mutation affected the expression of callose synthesis genes (CALLOSE SYNTHASE5 and CALLOSE SYNTHASE12) and potential callase-related genes (A6 and MYB80), as well as three other putative ß-1,3-glucanase genes. Therefore, we propose that the CDM1 gene regulates callose metabolism during microsporogenesis, thereby promoting Arabidopsis male fertility.

Evaluation of Streptomyces spp. for their plant-growth-promotion traits in rice.

Five strains of Streptomyces (CAI-17, CAI-68, CAI-78, KAI-26, and KAI-27) were previously reported to have potential for charcoal rot control and plant growth promotion (PGP) in sorghum. In this study, those 5 Streptomyces strains were characterized for their enzymatic activities and evaluated for their PGP capabilities on rice. All the Streptomyces strains were able to produce lipase and ß-1,3-glucanase; grew in NaCl (up to 8%), at pH 5-13, and at temperatures 20-40 °C; and were resistant to ampicillin, sensitive to nalidixic acid, and highly sensitive to chloramphenicol, kanamycin, streptomycin, and tetracycline. They were highly tolerant to the fungicide bavistin but were highly sensitive to benlate, benomyl, and radonil. When evaluated on rice in the field, Streptomyces significantly enhanced tiller and panicle numbers, stover and grain yields, dry matter, root length, volume and dry weight, compared with the control. In the rhizosphere at harvest, microbial biomass carbon and nitrogen, dehydrogenase activity, total nitrogen, available phosphorus, and % organic carbon were also found significantly higher in Streptomyces-treated plots than in the control plots. This study further confirms that the selected Streptomyces have PGP activities.

Comparative in vitro and in planta analyses of extracellular enzymes secreted by the pathogenic fungus Sclerotinia sclerotiorum.

Dry bean (Phaseolus vulgaris L.) is an important economic crop in Brazil but its yield can be significantly reduced by white mold, a disease caused by Sclerotinia sclerotiorum (Lib.) de Bary, a necrotrophic, highly destructive, and non-host-specific fungus. This fungus secretes numerous cell wall-degrading enzymes such as polygalacturonases, exo-ß-1,3-glucanases, xylanases, and cellulases that have been detected during the early stages of infection. In this study, the activities of these enzymes were detected in all carbon sources tested (citrus pectin, cell wall extract from P. vulgaris, carboxymethyl cellulose, and glucose), but the highest levels were found when using citrus pectin and cell wall extract from P. vulgaris. Regardless of the carbon source, pH decreased throughout the culture time. During pathogenesis in dry bean stems, increased enzyme activities were also observed. Reverse transcriptase-polymerase chain reaction experiments showed that the induction of polygalacturonases (sspg1, sspg3, sspg5, sspg6, and sspg7), exo-ß-1,3-glucanases, and endo-ß-1-4-glucanase in S. sclerotiorum occurred during the early stages of colonization.

Influence of the fungus Trichoderma harzianum on the enzyme and polysaccharide composition of Silene vulgaris Callus.

Activities of polygalacturonase and 1,3-ß-glucanase increased in campion (Silene vulgaris) callus cells during co-cultivation with the fungus Trichoderma harzianum. This was associated with a decrease in galacturonic acid residues in the pectic polysaccharide of campion silenan and also in the production of pectin by the callus. Co-cultivation of the callus and the fungus resulted in an increase in contents of arabinose residues in the intracellular arabinogalactan and in contents of galactose residues in the extracellular arabinogalactan.

Structures of an active-site mutant of a plant 1,3-ß-glucanase in complex with oligosaccharide products of hydrolysis.

Plant endo-1,3-ß-glucanases are involved in important physiological processes such as defence mechanisms, cell division and flowering. They hydrolyze (1→3)-ß-glucans, with very limited activity towards mixed (1→3,1→4)-ß-glucans and branched (1→3,1→6)-ß-glucans. Here, crystal structures of the potato (Solanum tuberosum) endo-1,3-ß-glucanase GLUB20-2 with the nucleophilic Glu259 residue substituted by alanine (E259A) are reported. Despite this active-site mutation, the protein retained residual endoglucanase activity and when incubated in the crystallization buffer with a linear hexameric substrate derived from (1→3)-ß-glucan (laminarahexose) cleaved it in two different ways, generating trisaccharides and tetrasaccharides, as confirmed by mass spectrometry. The trisaccharide (laminaratriose) shows higher binding affinity and was found to fully occupy the -1, -2 and -3 sites of the active-site cleft, even at a low molar excess of the substrate. At elevated substrate concentration the tetrasaccharide molecule (laminaratetrose) also occupies the active site, spanning the opposite sites +1, +2, +3 and +4 of the cleft. These are the first crystal structures of a plant glycoside hydrolase family 17 (GH17) member to reveal the protein-saccharide interactions and were determined at resolutions of 1.68 and 1.55 Å, respectively. The geometry of the active-site cleft clearly precludes any (1→4)-ß-glucan topology at the subsites from -3 to +4 and could possibly accommodate ß-1,6-branching only at subsites +1 and +2. The glucose units at subsites -1 and -2 interact with highly conserved protein residues. In contrast, subsites -3, +3 and +4 are variable, suggesting that the mode of glucose binding at these sites may vary between different plant endo-1,3-ß-glucanases. Low substrate affinity is observed at subsites +1 and +2, as manifested by disorder of the glycosyl units there.

Pollen wall development: the associated enzymes and metabolic pathways.

Pollen grains are surrounded by a sculpted wall, which protects male gametophytes from various environmental stresses and microbial attacks, and also facilitates pollination. Pollen wall development requires lipid and polysaccharide metabolism, and some key genes and proteins that participate in these processes have recently been identified. Here, we summarise the genes and describe their functions during pollen wall development via several metabolic pathways. A working model involving substances and catalytic enzyme reactions that occur during pollen development is also presented. This model provides information on the complete process of pollen wall development with respect to metabolic pathways.

Characterization of callase (ß-1,3-D-glucanase) activity during microsporogenesis in the sterile anthers of Allium sativum L. and the fertile anthers of A. atropurpureum.

We examined callase activity in anthers of sterile Allium sativum (garlic) and fertile Allium atropurpureum. In A. sativum, a species that produces sterile pollen and propagates only vegetatively, callase was extracted from the thick walls of A. sativum microspore tetrads exhibited maximum activity at pH 4.8, and the corresponding in vivo values ranged from 4.5 to 5.0. Once microspores were released, in vitro callase activity peaked at three distinct pH values, reflecting the presence of three callase isoforms. One isoform, which was previously identified in the tetrad stage, displayed maximum activity at pH 4.8, and the remaining two isoforms, which were novel, were most active at pH 6.0 and 7.3. The corresponding in vivo values ranged from pH 4.75 to 6.0. In contrast, in A. atropurpureum, a sexually propagating species, three callase isoforms, active at pH 4.8-5.2, 6.1, and 7.3, were identified in samples of microsporangia that had released their microspores. The corresponding in vivo value for this plant was 5.9. The callose wall persists around A. sativum meiotic cells, whereas only one callase isoform, with an optimum activity of pH 4.8, is active in the acidic environment of the microsporangium. However, this isoform is degraded when the pH rises to 6.0 and two other callase isoforms, maximally active at pH 6.0 and 7.3, appear. Thus, factors that alter the pH of the microsporangium may indirectly affect the male gametophyte development by modulating the activity of callase and thereby regulating the degradation of the callose wall.

Effects of genetic modifications to flax (Linum usitatissimum) on arbuscular mycorrhiza and plant performance.

Although arbuscular mycorrhizal fungi (AMF) are known for their positive effect on flax growth, the impact of genetic manipulation in this crop on arbuscular mycorrhiza and plant performance was assessed for the first time. Five types of transgenic flax that were generated to improve fiber quality and resistance to pathogens, through increased levels of either phenylpropanoids (W92.40), glycosyltransferase (GT4, GT5), or PR2 beta-1,3-glucanase (B14) or produce polyhydroxybutyrate (M50), were used. Introduced genetic modifications did not change the degree of mycorrhizal colonization as compared to parent cultivars Linola and Nike. Arbuscules were well developed in each tested transgenic type (except M50). In two lines (W92.40 and B14), a higher abundance of arbuscules was observed when compared to control, untransformed flax plants. However, in some cases (W92.40, GT4, GT5, and B14 Md), the mycorrhizal dependency for biomass production of transgenic plants was slightly lower when compared to the original cultivars. No significant influence of mycorrhiza on the photosynthetic activity of transformed lines was found, but in most cases P concentration in mycorrhizal plants remained higher than in nonmycorrhizal ones. The transformed flax lines meet the demands for better quality of fiber and higher resistance to pathogens, without significantly influencing the interaction with AMF.

LL37 and hBD-3 elevate the ß-1,3-exoglucanase activity of Candida albicans Xog1p, resulting in reduced fungal adhesion to plastic.

The opportunistic fungus Candida albicans causes oral thrush and vaginal candidiasis, as well as candidaemia in immunocompromised patients including those undergoing cancer chemotherapy, organ transplant and those with AIDS. We previously found that the AMPs (antimicrobial peptides) LL37 and hBD-3 (human ß-defensin-3) inhibited C. albicans viability and its adhesion to plastic. For the present study, the mechanism by which LL37 and hBD-3 reduced C. albicans adhesion was investigated. After AMP treatment, C. albicans adhesion to plastic was reduced by up to ~60% and was dose-dependent. Our previous study indicated that LL37 might interact with the cell-wall ß-1,3-exoglucanase Xog1p, which is involved in cell-wall ß-glucan metabolism, and consequently the binding of LL37 or hBD-3 to Xog1p might cause the decrease in adhesion. For the present study, Xog1p(41-438)-6H, an N-terminally truncated, active, recombinant construct of Xog1p and Xog1p fragments were produced and used in pull-down assays and ELISA in vitro, which demonstrated that all constructs interacted with both AMPs. Enzymatic analyses showed that LL37 and hBD-3 enhanced the ß-1,3-exoglucanase activity of Xog1p(41-438)-6H approximately 2-fold. Therefore elevated Xog1p activity might compromise cell-wall integrity and decrease C. albicans adhesion. To test this hypothesis, C. albicans was treated with 1.3 µM Xog1p(41-438)-6H and C. albicans adhesion to plastic decreased 47.7%. Taken together, the evidence suggests that Xog1p is one of the LL37/hBD-3 targets, and elevated ß-1,3-exoglucanase activity reduces C. albicans adhesion to plastic.

Abscisic acid enhances resistance to Alternaria solani in tomato seedlings.

The plant hormone abscisic acid (ABA) is an important regulator in many aspects of plant growth and development, as well as stress resistance. Here, we investigated the effects of exogenous ABA application on the interaction between tomato (Solanum lycopersicon L.) and Alternaria solani (early blight). Foliar spraying of 7.58 µM ABA was effective in reducing disease severity in tomato plants. Previously, increased activities of phenylalanine ammonia-lyase (PAL), polyphenol oxidase (PPO) and peroxidase (POD) were observed in exogenous ABA-treated tomato leaves. Moreover, these enzyme activities were maintained at higher levels in ABA-pretreated and A. solani challenged tomato plants. Tomato defense genes, such as PR1, ß-1, 3-glucanase (GLU), PPO, POD, and superoxide dismutase (SOD), were rapidly and significantly up-regulated by exogenous ABA treatment. Furthermore, a subsequent challenge of ABA-pretreated plants with the pathogen A. solani resulted in higher expression of defense genes, compared to water-treated or A. solani inoculated plants. Therefore, our results suggest that exogenous ABA could enhance disease resistance against A. solani infection in tomato through the activation of defense genes and via the enhancement of defense-related enzymatic activities.

Induction of systemic resistance in rice by leaf extracts of Zizyphus jujuba and Ipomoea carnea against Rhizoctonia solani.

Plants accumulate a great diversity of natural products, many of which confer protective effects against phytopathogenic attack. Earlier we had demonstrated that the leaf extracts of Zizyphus jujuba and Ipomoea carnea inhibit the in vitro mycelial growth of Rhizoctonia solani, and effectively reduce the incidence of sheath blight disease in rice. Here we demonstrate that foliar application of the aqueous leaf extracts of Z. jujuba and I. carnea followed by challenge inoculation with R. solani induces systemic resistance in rice as evident from significantly increased accumulation of pathogenesis-related proteins such as chitinase, ß-1,3-glucanase and peroxidase, as well as defense-related compounds such as phenylalanine ammonia-lyase and phenolic substances. Thin layer chromatographic separation of secondary metabolites revealed presence of alkaloid and terpenoid compounds in the leaf extracts of Z. jujuba that exhibited toxicity against R. solani under in vitro condition. Thus, the enhanced sheath blight resistance in rice seedlings treated with leaf extracts of Z. jujuba or I. carnea can be attributed to the direct inhibitory effects of these leaf extracts as well as their ability to elicit systemic resistance against R. solani.

Use of UV-C treatment to inhibit the microbial growth and maintain the quality of Yali pear.

UNLABELLED: The effects of UV-C radiation on microbial growth in vitro (Monilinia fruticola) and in inoculated Yali pears (Pyrus bretschneideri Rehd.) were investigated. Moreover, postharvest quality and the activities of defense and antioxidant enzymes were analyzed after the pears were exposed to UV-C irradiation at an energy level of 5 kJ m⁻².The results showed that spore germination of M. fructicola was significantly inhibited by each of the 3 doses (1, 5, and 10 kJ m⁻²) in vitro. In the in vivo assays, lesion diameter on the fruit being inoculated before or after the UV-C treatment was both significantly lower than that on the fruit of control. Meanwhile, the activities of phenylalanine ammonia lyase, ß-1,3-glucanase, superoxide dismutase, catalase, and glutathione reductase were induced to high levels by UV-C treatment. We conclude that UV-C treatment could reduce postharvest disease by the germicidal and induced effects and maintain the quality by enhancing the antioxidant enzyme activities. PRACTICAL APPLICATION: UV-C radiation has recently been proposed as a new technology to avoid chemical fungicides. However, there are few studies regarding the effect of UV-C treatment on Yali pear. In this study, we found that 5 kJ m⁻² UV-C irradiation can control postharvest disease and maintain the quality of Yali pear. This method may be applied to reduce the decay of Yali pears during exporting and storage.

[Agrobacterium tumefaciens mediated Chitinase and beta-1,3-glucanase gene transformation for Pinellia ternata].

OBJECTIVE: To obtain transgenic Pinellia ternata plants resistant to fungus by transfer Chitinase and beta-1,3-Glucanase gene from Trichoderma harzianum. METHOD: Using hygromycin phosphotransferase as the selection marker, the Chitinase gene (ech42), beta-1,3-Glucanase gene (gluc78) and both gene pCAMBIA(ech42 + gluc78) driven by CaMV35S promoter were transferred into P. ternata callus via Agrobacterium-mediated transformation. RESULT: PCR results confirmed that the regenerants were identified to be transgenic lines and the RT-PCR results confirmed that foreign genes construction were transfer to mRNA. Two foreign genes were inherited stably to T5 generation according to PCR results of the lines. CONCLUSION: The results showed that chitinase gene ech42 and beta-1, 3-glucanase gene gluc78 respectively or together introducing and co-integrating into P. ternata