Protein Elicitor Research Articles

Microtubules and biotic interactions

Plant microtubules undergo extensive reorganization in response to symbiotic and pathogenic organisms. During the development of successful symbioses with rhizobia and mycorrhizal fungi, novel microtubule arrays facilitate the progression of infection threads and hyphae, respectively, from the plant surface through epidermal and cortical cells. During viral and nematode infections, plant microtubules appear to be commandeered by the pathogen. Viruses use plant microtubules for intra and intercellular movement, as well as for interhost transmission. Nematodes manipulate spindle and phragmoplast microtubules to enhance mitosis and partial cytokinesis during the development of syncytia and giant cells. Pathogenic bacteria, fungi and oomycetes induce a range of alterations to microtubule arrays and dynamics. In many situations, the pathogen, or the elicitor or effector proteins derived from them, induce depolymerization of plant cortical microtubule arrays. In some cases, microtubule disruption is associated with the plant defence response and resistance. In other cases, microtubule depolymerization increases plant susceptibility to the invading pathogen. The reasons for this apparent inconsistency may depend on a number of factors, in particular on the identity of the organism orchestrating the microtubule changes. Overall, the weight of evidence indicates that microtubules play an important role in both the establishment of functional symbioses and in defence against invading pathogens. Research is beginning to unravel details about the nature of both the chemical and the mechanical signals to which the plant microtubule arrays respond during biotic interactions.

Interaction between abscisic acid and nitric oxide in PB90‐induced catharanthine biosynthesis of catharanthus roseus cell suspension cultures

Elicitations are considered to be an important strategy to improve production of secondary metabolites of plant cell cultures. However, mechanisms responsible for the elicitor-induced production of secondary metabolites of plant cells have not yet been fully elucidated. Here, we report that treatment of Catharanthus roseus cell suspension cultures with PB90, a protein elicitor from Phytophthora boehmeriae, induced rapid increases of abscisic acid (ABA) and nitric oxide (NO), subsequently followed by the enhancement of catharanthine production and up-regulation of Str and Tdc, two important genes in catharanthine biosynthesis. PB90-induced catharanthine production and the gene expression were suppressed by the ABA inhibitor and NO scavenger respectively, showing that ABA and NO are essential for the elicitor-induced catharanthine biosynthesis. The relationship between ABA and NO in mediating catharanthine biosynthesis was further investigated. Treatment of the cells with ABA triggered NO accumulation and induced catharanthine production and up-regulation of Str and Tdc. ABA-induced catharanthine production and gene expressions were suppressed by the NO scavenger. Conversely, exogenous application of NO did not stimulate ABA generation and treatment with ABA inhibitor did not suppress NO-induced catharanthine production and gene expressions. Together, the results showed that both NO and ABA were involved in PB90-induced catharanthine biosynthesis of C. roseus cells. Furthermore, our data demonstrated that ABA acted upstream of NO in the signaling cascade leading to PB90-induced catharanthine biosynthesis of C. roseus cells.

Purification and Characterization of AsES Protein: A SUBTILISIN SECRETED BY ACREMONIUM STRICTUM IS A NOVEL PLANT DEFENSE ELICITOR

In this work, the purification and characterization of an extracellular elicitor protein, designated AsES, produced by an avirulent isolate of the strawberry pathogen Acremonium strictum, are reported. The defense eliciting activity present in culture filtrates was recovered and purified by ultrafiltration (cutoff, 30 kDa), anionic exchange (Q-Sepharose, pH 7.5), and hydrophobic interaction (phenyl-Sepharose) chromatographies. Two-dimensional SDS-PAGE of the purified active fraction revealed a single spot of 34 kDa and pI 8.8. HPLC (C2/C18) and MS/MS analysis confirmed purification to homogeneity. Foliar spray with AsES provided a total systemic protection against anthracnose disease in strawberry, accompanied by the expression of defense-related genes (i.e. PR1 and Chi2-1). Accumulation of reactive oxygen species (e.g. H2O2 and O2(˙)) and callose was also observed in Arabidopsis. By using degenerate primers designed from the partial amino acid sequences and rapid amplification reactions of cDNA ends, the complete AsES-coding cDNA of 1167 nucleotides was obtained. The deduced amino acid sequence showed significant identity with fungal serine proteinases of the subtilisin family, indicating that AsES is synthesized as a larger precursor containing a 15-residue secretory signal peptide and a 90-residue peptidase inhibitor I9 domain in addition to the 283-residue mature protein. AsES exhibited proteolytic activity in vitro, and its resistance eliciting activity was eliminated when inhibited with PMSF, suggesting that its proteolytic activity is required to induce the defense response. This is, to our knowledge, the first report of a fungal subtilisin that shows eliciting activity in plants. This finding could contribute to develop disease biocontrol strategies in plants by activating its innate immunity.

Indução de resistência à podridão‑amarga em maçãs pelo uso de eliciadores em pós‑colheita

O objetivo deste trabalho foi avaliar o efeito dos eliciadores acibenzolar‑S‑metílico (ASM) e proteína harpina, aplicados em pós‑colheita, na indução de resistência sistêmica à podridão‑amarga em maçãs. Realizaram-se ferimentos mecânicos em maçãs 'Royal Gala' seguidos da aplicação dos eliciadores. Doze horas depois, procedeu-se à inoculação do fungo Colletotrichum gloeosporioides. Após 72 horas, realizaram-se as avaliações quanto à área lesionada e ao número de esporos, bem como a coleta de tecido dos frutos para quantificação de proteínas, açúcares totais e redutores, fenóis totais, e para determinação da atividade das enzimas fenilalanina amônia‑liase, superóxido dismutase, catalase, peroxidase e ascorbato peroxidase. A harpina e, em menor grau, o ASM proporcionaram aumento da atividade da enzima peroxidase e a consequente redução da área lesionada e da esporulação de C. gloeosporioides nas maçãs. Esses eliciadores podem ser utilizados como ferramenta de controle no manejo integrado da podridão‑amarga, em pós‑colheita de maçãs 'Royal Gala'.

Early transcription of defence-related genes in Platanus × acerifolia leaves following treatment with cerato-platanin

The protein elicitor cerato-platanin (CP) is known to induce defence-related responses in various plants. Some of these responses occur very quickly. In the present work, transcriptional changes caused by CP in leaves from Platanus × acerifolia (Aiton) Willd. were studied. With a cDNA microarray, 131 differentially regulated transcripts were identified as responsive to CP after 24 h of treatment. Eighty-six of these were cold-or ozone-modulated transcripts, thus revealing a significant overlap between genes responsive to CP and to cold/ozone stress. The transcriptional changes caused by CP were compared with the CP-orthologous protein Pop1 in a time-course analysis performed after 3, 6, 12, and 24 h of treatment by real-time RT-PCR on five defence-related genes. Despite some differences, CP and Pop1 were both able to induce early transcriptional changes (WRKY was overexpressed after only 3 h) confirming that pathogenassociated molecular patterns (PAMPs) act very quickly on gene transcription.

Overexpression of Elicitor Protein Encoding Gene hrip1 Improves Tolerance to Drought and Salt Stresses in Arabidopsis

hrip1 is a novel hypersensitive response-inducing protein secreted by necrotrophic fungus,Alternaria tenuissima.The Arabidopsis thaliana transgenic plants overexpressing hrip1 showed increased tolerance to drought and salt stresses.Five transformed lines of Arabidopsis were confirmed by PCR,RT-PCR and Western blot analysis.T4 progenies of transgenic Arabidopsis lines and the wild-type plants were tested.The results showed the seed germination rate and root length of transgenic plants were all significantly higher than those of wild type plants(P0.05) under stress conditions.The average seed relative germination rate of transgenic Arabidopsis lines was 32.1% and 77.9%,which were 3.72 and 5.61 times higher than those of wild-type plants in the two days incubation with 50 mmol L 1 NaCl and 75 mmol L 1 mannitol,respectively.The average relative root length in transgenic plants was 81.79% and 93.25%,which were 1.53 and 1.34 times of that in wild type plants at 7th day on the medium containing 150 mmol L 1 NaCl and 50 mmol L 1mannitol,respectively.Stress tolerances were improved in the three-week old soil-grown seedlings.Survival rate of transgenic plants and wild type plants was 67 % and 42 % under 250 mmol L 1NaCl stress for 20 days,respectively and 72 % of the transgenic plants and 44 % of wild-type plants were survived at 5th day re-watering after dehydration stress for 25 days.Otherwise,POD and CAT activities were increased in transgenic plants compared with three wild type plants under salt and drought stresses.At 24 h of treatment with 200 mmol L 1NaCl and 200 mmol L 1mannitol,the average POD activity in transgenic seedlings was 1.56 and 1.85 times,CAT activity was 1.64 and 1.86 times higher than those in wild plants,respectively.These data indicated that the expression of hrip1 gene positively regulates salt and drought tolerance in transgenic Arabidopsis.

Shape evolution with temperature of a thermotolerant protein (PeaT1) in solution detected by small angle X‐ray scattering

The protein elicitor from Alternaria tenuissima (PeaT1) presented excellent thermotolerance and potential application in agriculture as a pesticide. Previous synchrotron radiation circular dichroism study demonstrated that the secondary structures in PeaT1 protein are reversible with temperature change. To further clarify the mechanism of its thermotolerance, synchrotron radiation small angle x-ray scattering (SAXS) technique was used to study the shape change of PeaT1 protein with temperature in this article. Ab initio structure restorations based on the SAXS data revealed that PeaT1 protein has a prolate shape with a P₂ symmetry axis along the prolate anisometric direction. With temperature increase, a gooseneck vase-like (25°C), to jug-like (55°C), then to oval (85°C) shape change can be found, and these shape changes are also approximately reversible with temperature decrease. PeaT1 protein contains two homogenous molecules, and each of them consists of F, NAC, T, and UBA domains. The structures of the four domains were predicted. Simulated annealing algorithm was used to superimpose the domain structures onto the SAXS shapes. It was found that all the structural domains have position rotation and translation with temperature change, but the NAC domains are relatively stable, playing a role of frame. This shape change information provides clues for further exploring its biological function and application.

Hrip1, a novel protein elicitor from necrotrophic fungus, Alternaria tenuissima, elicits cell death, expression of defence‐related genes and systemic acquired resistance in tobacco

Here, we report the identification, purification, characterization and gene cloning of a novel hypersensitive response inducing protein secreted by necrotrophic fungus, Alternaria tenuissima, designated as hypersensitive response inducing protein 1 (Hrip1). The protein caused the formation of necrotic lesions that mimic a typical hypersensitive response and apoptosis-related events including DNA laddering. The protein-encoding gene was cloned by rapid amplification of cDNA ends (RACE) method. The sequence analysis revealed that the cDNA is 495 bp in length and the open reading frame (ORF) encodes for a polypeptide of 163 amino acids with theoretical pI of 5.50 and molecular weight of 17 562.5 Da. Hrip1 induced calcium influx, medium alkalinization, activation of salicylic acid-induced protein kinase and several defence-related genes after infiltration in tobacco leaves. Cellular damage, restricted to the infiltrated zone, occurred only several hours later, at a time when expression of defence-related genes was activated. After several days, systemic acquired resistance was also induced. The tobacco plant cells that perceived the Hrip1 generated a cascade of signals acting at local, short, and long distances, and caused the coordinated expression of specific defence responses in a way similar to hypersensitivity to tobacco mosaic virus. Thus, Hrip1 represents a powerful tool to investigate further the signals and their transduction pathways involved in induced disease resistance in necrotrophic fungi.

Purification and Characterization of a Novel Hypersensitive Response-Inducing Elicitor from Magnaporthe oryzae that Triggers Defense Response in Rice

Background Magnaporthe oryzae, the rice blast fungus, might secrete certain proteins related to plant-fungal pathogen interactions.Methodology/Principal FindingsIn this study, we report the purification, characterization, and gene cloning of a novel hypersensitive response-inducing protein elicitor (MoHrip1) secreted by M. oryzae. The protein fraction was purified and identified by de novo sequencing, and the sequence matched the genomic sequence of a putative protein from M. oryzae strain 70-15 (GenBank accession No. XP_366602.1). The elicitor-encoding gene mohrip1 was isolated; it consisted of a 429 bp cDNA, which encodes a polypeptide of 142 amino acids with a molecular weight of 14.322 kDa and a pI of 4.53. The deduced protein, MoHrip1, was expressed in E. coli. And the expression protein collected from bacterium also forms necrotic lesions in tobacco. MoHrip1 could induce the early events of the defense response, including hydrogen peroxide production, callose deposition, and alkalization of the extracellular medium, in tobacco. Moreover, MoHrip1-treated rice seedlings possessed significantly enhanced systemic resistance to M. oryzae compared to the control seedlings. The real-time PCR results indicated that the expression of some pathogenesis-related genes and genes involved in signal transduction could also be induced by MoHrip1.Conclusion/SignificanceThe results demonstrate that MoHrip1 triggers defense responses in rice and could be used for controlling rice blast disease.

Identification and Characterization of Tomato Mutants Affected in theRx-Mediated Resistance to PVX Isolates

Five tomato mutants affected in the Rx-mediated resistance against Potato virus X (PVX) were identified by screening a mutagenized population derived from a transgenic, Rx1-expressing 'Micro-Tom' line. Contrary to their parental line, they failed to develop lethal systemic necrosis upon infection with the virulent PVX-KH2 isolate. Sequence analysis and quantitative reverse-transcription polymerase chain reaction experiments indicated that the mutants are not affected in the Rx1 transgene or in the Hsp90, RanGap1 and RanGap2, Rar1 and Sgt1 genes. Inoculation with the PVX-CP4 avirulent isolate demonstrated that the Rx1 resistance was still effective in the mutants. In contrast, the virulent PVX-KH2 isolate accumulation was readily detectable in all mutants, which could further be separated in two groups depending on their ability to restrict the accumulation of PVX-RR, a mutant affected at two key positions for Rx1 elicitor activity. Finally, transient expression of the viral capsid protein elicitor indicated that the various mutants have retained the ability to mount an Rx1-mediated hypersensitive response. Taken together, the results obtained are consistent with a modification of the specificity or intensity of the Rx1-mediated response. The five Micro-Tom mutants should provide very valuable resources for the identification of novel tomato genes affecting the functioning of the Rx gene.

Protein Elicitor PemG1 fromMagnaporthe griseaInduces Systemic Acquired Resistance (SAR) in Plants

Elicitors can stimulate defense responses in plants and have become a popular strategy in plant disease control. Previously, we isolated a novel protein elicitor, PemG1, from Magnaporthe grisea. In the present study, PemG1 protein expressed in and purified from Escherichia coli improved resistance of rice and Arabidopsis to bacterial infection, induced transient expression of pathogenesis-related (PR) genes, and increased accumulation of hydrogen peroxide in rice. The effects of PemG1 on disease resistance and PR gene expression were mobilized systemically throughout the rice plant and persisted for more than 28 days. PemG1-induced accumulation of OsPR-1a in rice was prevented by the calcium channel blockers LaCl₃, BAPTA, EGTA, W7, and TFP. Arabidopsis mutants that are insensitive to jasmonic acid (JA) and ethylene showed increased resistance to bacterial infection after PemG1 treatment but PemG1 did not affect resistance of mutants with an impaired salicylic acid (SA) transduction pathway. In rice, PemG1 induced overexpressions of the SA signal-related genes (OsEDS1, OsPAL1, and OsNH1) but not the JA pathway-related genes (OsLOX2 and OsAOS2). Our findings reveal that PemG1 protein can function as an activator of plant disease resistance, and the PemG1-mediated systemic acquired resistance is modulated by SA- and Ca(2+)-related signaling pathways.

HarpinPss-mediated enhancement in growth and biological control of late leaf spot in groundnut by a chlorothalonil-tolerant Bacillus thuringiensis SFC24

Chemical and biological approaches have been adopted to increase the growth and yield of crops and reduce loss due to diseases. We have adopted an integrated approach, where both direct antagonism and induced resistance were combined to reduce the incidence of late leaf spot (LLS) disease in groundnut caused by Phaeoisariopsis personata. Chitinolytic chlorothalonil-tolerant soil bacterium Bacillus thuringiensis SFC24 (Bt SFC24) was manipulated in vitro to express secretable form of elicitor protein harpinPss of Pseudomonas syringae pv. syringae. Severity of the LLS decreased by 65% when the leaves were sprayed with B. thuringiensis expressing harpinPss (Bt-pss). As seed treatment, there was an increase in growth of groundnut. Bt and Bt-pss accounted to 13% and 36% increase in shoot length. Expression of a secretable form of harpinPss thus improved the ability of B. thuringiensis SFC24 to promote growth and control LLS in groundnut. In this new approach a chlorothalonil-tolerant chitinolytic bacterium was genetically engineered to secrete elicitor harpinPss for dual benefit of growth promotion and disease control.

Phosphatidylinositol monophosphate-binding interface in the oomycete RXLR effector AVR3a is required for its stability in host cells to modulate plant immunity

The oomycete pathogen Phytophthora infestans causes potato late blight, one of the most economically damaging plant diseases worldwide. P. infestans produces AVR3a, an essential modular virulence effector with an N-terminal RXLR domain that is required for host-cell entry. In host cells, AVR3a stabilizes and inhibits the function of the E3 ubiquitin ligase CMPG1, a key factor in host immune responses including cell death triggered by the pathogen-derived elicitor protein INF1 elicitin. To elucidate the molecular basis of AVR3a effector function, we determined the structure of Phytophthora capsici AVR3a4, a close homolog of P. infestans AVR3a. Our structural and functional analyses reveal that the effector domain of AVR3a contains a conserved, positively charged patch and that this region, rather than the RXLR domain, is required for binding to phosphatidylinositol monophosphates (PIPs) in vitro. Mutations affecting PIP binding do not abolish AVR3a recognition by the resistance protein R3a but reduce its ability to suppress INF1-triggered cell death in planta. Similarly, stabilization of CMPG1 in planta is diminished by these mutations. The steady-state levels of non-PIP-binding mutant proteins in planta are reduced greatly, although these proteins are stable in vitro. Furthermore, overexpression of a phosphatidylinositol phosphate 5-kinase results in reduction of AVR3a levels in planta. Our results suggest that the PIP-binding ability of the AVR3a effector domain is essential for its accumulation inside host cells to suppress CMPG1-dependent immunity.

The purification and characterization of a novel hypersensitive-like response-inducing elicitor from Verticillium dahliae that induces resistance responses in tobacco

PevD1, a novel protein elicitor from the pathogenic cotton verticillium wilt fungus, Verticillium dahliae, induced a hypersensitive response in tobacco plants. In this paper, the elicitor was purified and analyzed using de novo sequencing. The protein-encoding pevD1 gene consists of a 468-bp open reading frame that produces a polypeptide of 155 amino acids, with a theoretical molecular weight of 16.23 kDa. The sequence of elicitor protein PevD1 was matched to the genomic sequence (GenBank accession no. ABJE 01000445.1) of a putative protein from V. dahliae strain vdls.17, but a function had not yet been reported. The pevD1 gene was expressed in Escherichia coli, and the recombinant protein was characterized for its ability to confer systemic acquired resistance to tobacco mosaic virus (TMV). Recombinant PevD1-treated plants exhibited enhanced systemic resistance compared to control, including a significant reduction in the number and size of TMV lesions on tobacco leaves. The elicitor protein-induced hydrogen peroxide production, extracellular-medium alkalization, callose deposition, phenolics metabolism, and lignin synthesis in tobacco. Our results demonstrate that elicitor-PevD1 triggers defense responses in intact tobacco plants.

Nitrate reductase-mediated nitric oxide generation is essential for fungal elicitor-induced camptothecin accumulation of Camptotheca acuminata suspension cell cultures

Secondary metabolite accumulation and nitric oxide (NO) generation are two common responses of plant cells to fungal elicitors, and NO has been reported to play important roles in elicitor-induced secondary metabolite production. However, the source of elicitor-triggered NO generation in plant cells remains largely unknown. To investigate the origin of elicitor-triggered NO, we examined nitrate reductase (NR) activities and the expression levels of NIA1 and NIA2 genes of Camptotheca acuminata cells treated with PB90, a protein elicitor from Phytophthora boehmeriae. The data show that PB90 treatment stimulates NR activity and induces upregulation of NIA1 but does not affect NIA2 expression in the cells. Pretreatment of the cells with NR inhibitors tungstate and Gln abolishes not only the fungal elicitor-triggered NR activities but also the PB90-induced NO generation. Treatment of PB90 enhances camptothecin contents of the cells, suggesting that the fungal elicitor might stimulate camptothecin biosynthesis. Furthermore, application of tungstate and Gln suppresses the fungal elicitor-induced camptothecin accumulation of the cells and the suppression of NR inhibitors on PB90-induced camptothecin production can be reversed by NO via its donor sodium nitroprusside. Together, the results suggest that NIA1 is sensitive to PB90 and the fungal elicitor-induced upregulation of NIA1 may lead to higher NR activity. Furthermore, our data demonstrate that NR is involved in the fungal elicitor-triggered NO generation and the fungal elicitor induces camptothecin production of C. acuminata cells dependently on NR-mediated NO generation.

Stable isotope labelled mass spectrometry for quantification of the relative abundances for expressed proteins induced by PeaT1

The protein elicitor from the mycelium of Alternaria tenuissima has been isolated. The elicitor triggered resistance to the tobacco mosaic virus in tobacco by inducing relative oxygen species, but without causing hypersensitive necrosis. The elicitor is reported to impart resistance against Verticillium dahliae and to increase yield in cotton, but its mechanism is not yet clear. In this study, the stable isotope labelled mass spectrometry method was used to quantify the relative abundances of protein expression induced by PeaT1 in Arabidopsis. A significant difference in the relative abundances for the expression of different proteins related to metabolism, modification, regulatory, defense, stress and antioxidation was found in Arabidopsis.

Involvement of NADPH oxidase-mediated H2O2 signaling in PB90-induced hypericin accumulation in Hypericum perforatum cells

PB90 is a novel protein elicitor isolated from Phytophthora boehmeriae. Here, we report that treatment of PB90 stimulates hypericin production and hydrogen peroxide (H2O2) generation in Hypericum perforatum L. cells and demonstrate that H2O2 is essential for PB90-induced hypericin production. To further study the source of PB90-triggered H2O2, we have investigated activities of plasma membrane NADPH oxidase in Hypericum perforatum L. cells subjected to PB90 treatment. It is revealed that treatment of the cells with PB90 significantly increases NADPH oxidase activity. NADPH oxidase inhibitors suppress not only the PB90-stimulated NADPH oxidase activity but also the PB90-triggered H2O2 generation and PB90-induced hypericin production, showing that NADPH oxidase is involved in PB90-triggered H2O2 generation and hypericin production. Moreover, the suppression of NADPH oxidase inhibitors on PB90-induced hypericin production can be reversed by H2O2, although H2O2 per se has no effects on hypericin production of the cells. Together, the data demonstrate that PB90 may induce hypericin production of H. perforatum cells through the NADPH oxidase-mediated H2O2 signaling pathway.

Cerato-platanin elicits transcription of defence-related genes earlier than Ceratocystis platani on Platanus acerifolia

The ascomycete Ceratocystis platani (Walter) Engelbrecht & Harrington is the causal agent of canker stain, the most dangerous disease affecting the plane tree. Cerato-platanin (CP) is a protein elicitor from C. platani that acts as a pathogen-associated molecular pattern (PAMP), eliciting defence responses in both host and non-host plants. Using plane leaves as an experimental model, we compared the capacity of CP and C. platani to cause alterations in the transcription level of five Platanus acerifolia (Ait.) Willd. genes, three strictly related to a defence response, and two non-defence related. The results suggest that CP elicits the transcription of defence-related genes earlier than does C. platani. The expression of the cp gene in C. platani inoculated on plane leaves and grown in an axenic culture was also studied. For the first time CP has been shown to be released by C. platani when inoculated on P. acerifolia. The cp gene was expressed sooner when C. platani was inoculated on the leaves, than when it was grown in axenic culture, suggesting that the host-tree has a positive effect on the expression of this elicitor protein.

Purification and expression of a protein elicitor from Alternaria tenuissima and elicitor‐mediated defence responses in tobacco

A new protein elicitor, PeaT1, was purified from the mycelium of Alternaria tenuissima by column chromatography. PeaT1 was identified as a heat‐stable and acidic protein. It induced systemic acquired resistance to tobacco mosaic virus (TMV) in tobacco plants but did not cause hypersensitive response. The elicitor‐encoding gene was cloned by rapid amplification of cDNA ends method. Sequence analysis revealed that the cDNA is 624 bp in length and the open reading frame encodes for a polypeptide of 207 amino acids with a nascent polypeptide‐associated complex domain. The peaT1 gene was cloned into the expression vector pET‐28a and transformed into Escherichia coli BL21 (DE3). The recombinant elicitor also triggered defence responses in intact tobacco plants. The availability of the pure protein offers the possibility to isolate the corresponding receptor and links it to the downstream signalling pathway.

Significance of protein elicitor isolated from Tuber melanosporum on the production of ganoderic acid and Ganoderma polysaccharides during the fermentation of Ganoderma lucidum

Under the elicitation of protein elicitor isolated from the culture mycelia of Tuber melanosporum, the biosynthesis of ganoderic acids (GA) was significantly stimulated during Ganoderma lucidum fermentation. Compared with our previous results that, GA content was inhibited by polysaccharide elicitor isolated from T. melanosporum, while improved by the elicitor of polysaccharide and protein, protein was identified to be the exact component inducing GA biosynthesis in this work. G. lucidum cell growth was significantly inhibited by elicitor of polysaccharide and protein, and polysaccharide elicitor did not inhibit the cell growth. In this work, the remarkable inhibition on the cell growth was considerably eliminated under the elicitation of protein elicitor isolated from T. melanosporum. These suggested maybe the interaction of polysaccharide and protein components existed in the inhibition on the cell growth of G. lucidum. Not only GA content but also total GA accumulation obtained the highest values after the elicitation of protein elicitor. The maximal GA production of 260.5 ± 5.6 mg/L was 31.2% higher than the control. Under the elicitation of protein elicitor, the production of extracellular polysaccharide (EPS) and the content of intracellular polysaccharide (IPS) were also enhanced; however, total IPS accumulation was lower. GA biosynthesis was also significantly affected by the addition time of protein elicitor, whose optimal value was the culture of day 4.