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1.
Biotechnol Lett ; 39(7): 1049-1058, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28365881

RESUMO

OBJECTIVES: To engineer broad spectrum resistance in potato using different expression strategies. RESULTS: The previously identified Ribosome-Inactivating Protein from Phytolacca heterotepala was expressed in potato under a constitutive or a wound-inducible promoter. Leaves and tubers of the plants constitutively expressing the transgene were resistant to Botrytis cinerea and Rhizoctonia solani, respectively. The wound-inducible promoter was useful in driving the expression upon wounding and fungal damage, and conferred resistance to B. cinerea. The observed differences between the expression strategies are discussed considering the benefits and features offered by the two systems. CONCLUSIONS: Evidence is provided of the possible impact of promoter sequences to engineer BSR in plants, highlighting that the selection of a suitable expression strategy has to balance specific needs and target species.


Assuntos
Resistência à Doença , Expressão Gênica , Organismos Geneticamente Modificados/imunologia , Doenças das Plantas/prevenção & controle , Proteínas Recombinantes/metabolismo , Proteínas Inativadoras de Ribossomos/metabolismo , Solanum tuberosum/imunologia , Botrytis/imunologia , Botrytis/patogenicidade , Regulação da Expressão Gênica de Plantas , Organismos Geneticamente Modificados/genética , Phytolacca/enzimologia , Phytolacca/genética , Doenças das Plantas/microbiologia , Proteínas Recombinantes/genética , Rhizoctonia/imunologia , Rhizoctonia/patogenicidade , Proteínas Inativadoras de Ribossomos/genética , Solanum tuberosum/genética
2.
World J Microbiol Biotechnol ; 30(4): 1229-38, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24197785

RESUMO

Rice sheath blight and blast caused by Rhizoctonia solani Kühn and Magnorpathe oryzae respectively, are the two most destructive fungal diseases in rice. With no genetic natural traits conferring resistance to sheath blight, transgenic manipulation provides an obvious approach. In this study, the rice basic chitinase gene (RCH10) and the alfalfa ß-1,3-glucanase gene (AGLU1) were tandemly inserted into transformation vector pBI101 under the control of 35S promoter with its enhancer sequence to generate a double-defense gene expression cassette pZ100. The pZ100 cassette was transformed into rice (cv. Taipei 309) by Agrobacterium-mediated transformation. More than 160 independent transformants were obtained and confirmed by PCR. Northern analysis of inheritable progenies revealed similar levels of both RCH10 and AGLU1 transcripts in the same individuals. Disease resistance to both sheath blight and blast was challenged in open field inoculation. Immunogold detection revealed that RCH10 and AGLU1 proteins were initially located mainly in the chloroplasts and were delivered to the vacuole and cell wall upon infection, suggesting that these subcellular compartments act as the gathering and execution site for these anti-fungal proteins. We also observed that transgenic seeds display lower germination rate and seedling vigor, indicating that defense enhancement might be achieved at the expense of development.


Assuntos
Quitinases/metabolismo , Glucana 1,3-beta-Glucosidase/metabolismo , Magnaporthe/imunologia , Oryza/imunologia , Doenças das Plantas/imunologia , Plantas Geneticamente Modificadas/imunologia , Rhizoctonia/imunologia , Quitinases/genética , Expressão Gênica , Glucana 1,3-beta-Glucosidase/genética , Mutagênese Insercional , Oryza/microbiologia , Desenvolvimento Vegetal , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas/microbiologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transformação Genética
3.
Theor Appl Genet ; 126(5): 1257-72, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23423653

RESUMO

Sheath blight (SB), caused by Rhizoctonia solani kühn, is one of the most serious global rice diseases. No major resistance genes to SB have been identified so far. All discovered loci are quantitative resistance to rice SB. The qSB-11(LE) resistance quantitative trait locus (QTL) has been previously reported on chromosome 11 of Lemont (LE). In this study, we report the precise location of qSB-11 (LE) . We developed a near isogenic line, NIL-qSB11(TQ), by marker-assisted selection that contains susceptible allele(s) from Teqing (TQ) at the qSB-11 locus in the LE genetic background. NIL-qSB11(TQ) shows higher susceptibility to SB than LE in both field and greenhouse tests, suggesting that this region of LE contains a QTL contributing to SB resistance. In order to eliminate the genetic background effects and increase the accuracy of phenotypic evaluation, a total of 112 chromosome segment substitution lines (CSSLs) with the substituted segment specific to the qSB-11 (LE) region were produced as the fine mapping population. The genetic backgrounds and morphological characteristics of these CSSLs are similar to those of the recurrent parent LE. The donor TQ chromosomal segments in these CSSL lines contiguously overlap to bridge the qSB-11 (LE) region. Through artificial inoculation, all CSSLs were evaluated for resistance to SB in the field in 2005. For the recombinant lines, their phenotypes were evaluated in the field for another 3 years and during the final year were also evaluated in a controlled greenhouse environment, showing a consistent phenotype in SB resistance across years and conditions. After comparing the genotypic profile of each CSSL with its phenotype, we are able to localize qSB-11 (LE) to the region defined by two cleaved-amplified polymorphic sequence markers, Z22-27C and Z23-33C covering 78.871 kb, based on the rice reference genome. Eleven putative genes were annotated within this region and three of them were considered the most likely candidates. The results of this study will greatly facilitate the cloning of the genes responsible for qSB-11 (LE) and marker-assisted breeding to incorporate qSB-11 (LE) into other rice cultivars.


Assuntos
Mapeamento Cromossômico , Resistência à Doença/genética , Oryza/genética , Doenças das Plantas/genética , Locos de Características Quantitativas , Rhizoctonia/patogenicidade , Cromossomos de Plantas , DNA de Plantas/genética , Genes de Plantas , Ligação Genética , Marcadores Genéticos , Imunidade Inata/genética , Oryza/imunologia , Oryza/microbiologia , Fenótipo , Doenças das Plantas/microbiologia , Rhizoctonia/genética , Rhizoctonia/imunologia
4.
J Agric Food Chem ; 60(43): 10723-32, 2012 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-23046447

RESUMO

Plant interactions with microbial biocontrol agents are used as experimental models to understand resistance-related molecular adaptations of plants. In a hydroponic three-way interaction study, a novel Trichoderma harzianum ETS 323 mediated mechanism was found to induce resistance to Rhizoctonia solani infection in Brassica oleracea var. capitata plantlets. The R. solani challenge on leaves initiate an increase in lipoxygenase activity and associated hypersensitive tissue damage with characteristic "programmed cell death" that facilitate the infection. However, B. oleracea plantlets whose roots were briefly (6 h) colonized by T. harzianum ETS 323 developed resistance to R. solani infection through a significant reduction of the host hypersensitive tissue damage. The resistance developed in the distal leaf tissue was associated with the expression of a H(2)O(2)-inducible glutathione S-transferase (BoGST), which scavenges cytotoxic reactive electrophiles, and of a deoxycytidine deaminase (BoDCD), which modulates the host molecular expression and potentially neutralizes the DNA adducts and maintains DNA integrity. The cDNAs of BoGST and BoDCD were cloned and sequenced; their expressions were verified by reverse-transcription polymerase chain reaction analysis and were found to be transcriptionally activated during the three-way interaction.


Assuntos
Brassica/enzimologia , Glutationa Transferase/genética , Nucleosídeo Desaminases/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Rhizoctonia/fisiologia , Trichoderma/fisiologia , Brassica/genética , Brassica/imunologia , Brassica/microbiologia , Citidina Desaminase , Regulação da Expressão Gênica de Plantas , Glutationa Transferase/imunologia , Nucleosídeo Desaminases/imunologia , Doenças das Plantas/genética , Proteínas de Plantas/imunologia , Rhizoctonia/imunologia
5.
Plant Cell Physiol ; 53(6): 1017-32, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22492233

RESUMO

Rhizoctonia solani Kuhn causes sheath blight disease in rice, and genetic resistance against it is the most desirable characteristic. Current improvement efforts are based on analysis of polygenic quantitative trait loci (QTLs), but interpretation is limited by the lack of information on the changes in metabolic pathways. Our previous studies linked activation of the glycolytic pathway to enhanced generation of lignin in the phenylpropanoid pathway. The current studies investigated the regulation of glycolysis by examining the time course of changes in enzymatic activities and metabolite contents. The results showed that the activities of all glycolytic enzymes as well as fructose-6-phosphate (F-6-P), fructose-1,6-bisphosphate (F-1,6-P(2)), dihydroxyacetone phosphate (DHAP), glyceraldehyde-3-phosphate (GAP), 3-phosphoglycerate (3-PG), phosphoenolpyruvate (PEP) and pyruvate contents increased. These results combined with our previous findings that the expression of phosphoglucomutase (PGM), triosephosphate isomerase (TPI), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), enolase and pyruvate kinase (PK) increased after infection suggested that the additional establishment of glycolysis in the cytosol compartment occurred after infection. Further evidence for this was our recent findings that the increase in expression of the 6-phosphofructokinase (PFK) plastid isozyme Os06g05860 was accompanied by an increase in expression of three cytosolic PFK isozymes, i.e. Os01g09570, Os01g53680 and Os04g39420, as well as pyrophosphate-dependent phosphofrucokinase (PFP) isozymes Os08g25720 (α-subunit) and Os06g13810 (ß-subunit) in infected rice plants of the resistant line. The results also showed that the reactions catalysed by PFK/PFP, aldolase, GAPDH + phosphoglycerate kinase (PGK) and PK in leaf sheaths of R. solani-infected rice plants were non-equilibrium reactions in vivo. This study showed that PGM, phosphoglucose isomerase (PGI), TPI and phosphoglycerate mutase (PGmu) + enolase could be regulated through coarse control whereas, PFK/PFP, aldolase, GAPDH + PGK and PK could be regulated through coarse and fine controls simultaneously.


Assuntos
Glicólise , Interações Hospedeiro-Patógeno , Oryza/enzimologia , Rhizoctonia/patogenicidade , Citosol/enzimologia , Citosol/metabolismo , Fosfato de Di-Hidroxiacetona/genética , Fosfato de Di-Hidroxiacetona/metabolismo , Resistência à Doença , Ativação Enzimática , Frutosedifosfatos/genética , Frutosedifosfatos/metabolismo , Frutosefosfatos/genética , Frutosefosfatos/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Glucose-6-Fosfato Isomerase/genética , Glucose-6-Fosfato Isomerase/metabolismo , Gliceraldeído 3-Fosfato/genética , Gliceraldeído 3-Fosfato/metabolismo , Peróxido de Hidrogênio/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Oryza/genética , Oryza/imunologia , Oryza/microbiologia , Fosfoenolpiruvato/metabolismo , Fosfofrutoquinase-1/genética , Fosfofrutoquinase-1/metabolismo , Fosfoglucomutase/genética , Fosfoglucomutase/metabolismo , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ácido Pirúvico/metabolismo , Locos de Características Quantitativas , Rhizoctonia/imunologia , Fatores de Tempo
6.
Biochem Biophys Res Commun ; 419(4): 708-14, 2012 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-22386990

RESUMO

We earlier reported the mitogenic and immunostimulatory activities of Rhizoctonia bataticola lectin (RBL), purified from phytopathogenic fungus R. bataticola in human PBMC. The lectin demonstrates specificity towards glycoproteins containing complex N-glycans. Since CD45-protein tyrosine phosphatase that abundantly expresses N-glycans is important in T-cell signaling, the study aimed to investigate the involvement of CD45 in the immunomodulatory activities of RBL. Flowcytometry and confocal microscopy studies revealed that RBL exhibited binding to PBMC and colocalized with CD45. The binding was comparable in cells expressing different CD45 isoforms-RA, -RB and -RO. CD45 blocking antibody reduced the binding and proliferation of PBMC induced by RBL. CD45-PTPase inhibitor dephostatin inhibited RBL-induced proliferation, expression of CD25 and pZAP-70. RBL-induced secretion of Th1/Th2 cytokines were significantly inhibited in presence of dephostatin. Also, dephostatin blocked phosphorylation of p38MAPK and STAT-5 that was crucial for the biological functions of RBL. The study demonstrates the involvement of CD45-mediated signaling in RBL-induced PBMC proliferation and Th1/Th2 cytokine secretion through activation of p38MAPK and STAT-5.


Assuntos
Citocinas/metabolismo , Imunomodulação , Lectinas/imunologia , Antígenos Comuns de Leucócito/imunologia , Rhizoctonia/imunologia , Células Th1/imunologia , Células Th2/imunologia , Proliferação de Células , Células Cultivadas , Humanos , Leucócitos Mononucleares/imunologia , Fosforilação , Fator de Transcrição STAT5/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
7.
PLoS One ; 6(9): e24677, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21931807

RESUMO

BACKGROUND: Turfgrass species are agriculturally and economically important perennial crops. Turfgrass species are highly susceptible to a wide range of fungal pathogens. Dollar spot and brown patch, two important diseases caused by fungal pathogens Sclerotinia homoecarpa and Rhizoctonia solani, respectively, are among the most severe turfgrass diseases. Currently, turf fungal disease control mainly relies on fungicide treatments, which raises many concerns for human health and the environment. Antimicrobial peptides found in various organisms play an important role in innate immune response. METHODOLOGY/PRINCIPAL FINDINGS: The antimicrobial peptide - Penaeidin4-1 (Pen4-1) from the shrimp, Litopenaeus setiferus has been reported to possess in vitro antifungal and antibacterial activities against various economically important fungal and bacterial pathogens. In this study, we have studied the feasibility of using this novel peptide for engineering enhanced disease resistance into creeping bentgrass plants (Agrostis stolonifera L., cv. Penn A-4). Two DNA constructs were prepared containing either the coding sequence of a single peptide, Pen4-1 or the DNA sequence coding for the transit signal peptide of the secreted tobacco AP24 protein translationally fused to the Pen4-1 coding sequence. A maize ubiquitin promoter was used in both constructs to drive gene expression. Transgenic turfgrass plants containing different DNA constructs were generated by Agrobacterium-mediated transformation and analyzed for transgene insertion and expression. In replicated in vitro and in vivo experiments under controlled environments, transgenic plants exhibited significantly enhanced resistance to dollar spot and brown patch, the two major fungal diseases in turfgrass. The targeting of Pen4-1 to endoplasmic reticulum by the transit peptide of AP24 protein did not significantly impact disease resistance in transgenic plants. CONCLUSION/SIGNIFICANCE: Our results demonstrate the effectiveness of Pen4-1 in a perennial species against fungal pathogens and suggest a potential strategy for engineering broad-spectrum fungal disease resistance in crop species.


Assuntos
Agrostis/metabolismo , Agrostis/microbiologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Resistência à Doença/fisiologia , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/microbiologia , Agrostis/genética , Peptídeos Catiônicos Antimicrobianos/genética , Ascomicetos/imunologia , Ascomicetos/patogenicidade , Resistência à Doença/genética , Micoses/imunologia , Plantas Geneticamente Modificadas/genética , Rhizoctonia/imunologia , Rhizoctonia/patogenicidade
8.
Plant J ; 68(5): 777-87, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21793954

RESUMO

Plant 14-3-3 proteins regulate important cellular processes, including plant immune responses, through protein-protein interactions with a wide range of target proteins. In rice (Oryza sativa), the GF14e gene, which encodes a 14-3-3 protein, is induced during effector-triggered immunity (ETI) associated with pathogens such as Xanthomonas oryzae pv. oryzae (Xoo). To determine whether the GF14e gene plays a direct role in resistance to disease in rice, we suppressed its expression by RNAi silencing. GF14e suppression was correlated with the appearance of a lesion-mimic (LM) phenotype in the transgenic plants at 3 weeks after sowing. This indicates inappropriate regulation of cell death, a phenotype that is frequently associated with enhanced resistance to pathogens. GF14e-silenced rice plants showed high levels of resistance to a virulent strain of Xoo compared with plants that were not silenced. Enhanced resistance was correlated with GF14e silencing prior to and after development of the LM phenotype, higher basal expression of a defense response peroxidase gene (POX22.3), and accumulation of reactive oxygen species (ROS). In addition, GF14e-silenced plants also exhibit enhanced resistance to the necrotrophic fungal pathogen Rhizoctonia solani. Together, our findings suggest that GF14e negatively affects the induction of plant defense response genes, cell death and broad-spectrum resistance in rice.


Assuntos
Proteínas 14-3-3/imunologia , Resistência à Doença , Oryza/imunologia , Proteínas de Plantas/imunologia , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Morte Celular , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Peróxido de Hidrogênio/metabolismo , Oryza/genética , Oryza/metabolismo , Oryza/microbiologia , Peroxidase/genética , Peroxidase/metabolismo , Fenótipo , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/imunologia , Plantas Geneticamente Modificadas/metabolismo , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Rhizoctonia/imunologia , Rhizoctonia/patogenicidade , Fatores de Tempo , Xanthomonas/imunologia , Xanthomonas/patogenicidade
9.
Phytopathology ; 101(7): 828-38, 2011 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-21385012

RESUMO

Certain hypovirulent Rhizoctonia isolates effectively protect plants against well-known important pathogens among Rhizoctonia isolates as well as against other pathogens. The modes of action involved in this protection include resistance induced in plants by colonization with hypovirulent Rhizoctonia isolates. The qualifications of hypovirulent isolates (efficient protection, rapid growth, effective colonization of the plants, and easy application in the field) provide a significant potential for the development of a commercial microbial preparation for application as biological control agents. Understanding of the modes of action involved in protection is important for improving the various aspects of development and application of such preparations. The hypothesis of the present study is that resistance pathways such as systemic acquired resistance (SAR), induced systemic resistance (ISR), and phytoalexins are induced in plants colonized by the protective hypovirulent Rhizoctonia isolates and are involved in the protection of these plants against pathogenic Rhizoctonia. Changes in protection levels of Arabidopsis thaliana mutants defective in defense-related genes (npr1-1, npr1-2, ndr1-1, npr1-2/ndr1-1, cim6, wrky70.1, snc1, and pbs3-1) and colonized with the hypovirulent Rhizoctonia isolates compared with that of the wild type (wt) plants colonized with the same isolates confirmed the involvement of induced resistance in the protection of the plants against pathogenic Rhizoctonia spp., although protection levels of mutants constantly expressing SAR genes (snc1 and cim6) were lower than that of wt plants. Plant colonization by hypovirulent Rhizoctonia isolates induced elevated expression levels of the following genes: PR5 (SAR), PDF1.2, LOX2, LOX1, CORI3 (ISR), and PAD3 (phytoalexin production), which indicated that all of these pathways were induced in the hypovirulent-colonized plants. When SAR or ISR were induced separately in plants after application of the chemical inducers Bion and methyl jasmonate, respectively, only ISR activation resulted in a higher protection level against the pathogen, although the protection was minor. In conclusion, plant colonization with the protective hypovirulent Rhizoctonia isolates significantly induced genes involved in the SAR, ISR, and phytoalexin production pathways. In the studied system, SAR probably did not play a major role in the mode of protection against pathogenic Rhizoctonia spp.; however, it may play a more significant role in protection against other pathogens.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/imunologia , Doenças das Plantas/microbiologia , Rhizoctonia/patogenicidade , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Mutação , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Imunidade Vegetal , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/microbiologia , Reação em Cadeia da Polimerase em Tempo Real , Rhizoctonia/genética , Rhizoctonia/imunologia , Sesquiterpenos/metabolismo , Virulência , Fitoalexinas
10.
Funct Integr Genomics ; 11(1): 63-70, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21279533

RESUMO

Fusarium head blight (scab), primarily caused by Fusarium graminearum, is a devastating disease of wheat (Triticum aestivum L.) worldwide. Wheat sharp eyespot, mainly caused by Rhizoctonia cerealis, is one of the major diseases of wheat in China. The defensin RsAFP2, a small cyteine-rich antifungal protein from radish (Raphanus sativus), was shown to inhibit growth in vitro of agronomically important fungal pathogens, such as F. graminearum and R. cerealis. The RsAFP2 gene was transformed into Chinese wheat variety Yangmai 12 via biolistic bombardment to assess the effectiveness of the defensin in protecting wheat from the fungal pathogens in multiple locations and years. The genomic PCR and Southern blot analyses indicated that RsAFP2 was integrated into the genomes of the transgenic wheat lines and heritable. RT-PCR and Western blot proved that the RsAFP2 was expressed in these transgenic wheat lines. Disease tests showed that four RsAFP2 transgenic lines (RA1-RA4) displayed enhanced resistance to F. graminearum compared to the untransformed Yangmai 12 and the null-segregated plants. Assays on Q-RT-PCR and disease severity showed that the express level of RsAFP2 was associated with the enhanced resistance degree. Two of these transgenic lines (RA1 and RA2) also exhibited enhanced resistance to R. cerealis. These results indicated that the expression of RsAFP2 conferred increased resistance to F. graminearum and R. cerealis in transgenic wheat.


Assuntos
Defensinas/metabolismo , Fusarium/imunologia , Doenças das Plantas/imunologia , Plantas Geneticamente Modificadas/genética , Raphanus/metabolismo , Rhizoctonia/imunologia , Triticum/genética , Southern Blotting , Western Blotting , China , DNA de Plantas/genética , Defensinas/genética , Defensinas/imunologia , Fusarium/genética , Fusarium/metabolismo , Imunidade Inata , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas/imunologia , Plantas Geneticamente Modificadas/microbiologia , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA de Plantas/genética , Raphanus/genética , Rhizoctonia/genética , Rhizoctonia/metabolismo , Triticum/imunologia , Triticum/microbiologia
11.
J Plant Physiol ; 168(10): 1114-22, 2011 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-21269732

RESUMO

We examined basal defense responses and cytomolecular aspects of riboflavin-induced resistance (IR) in sugar beet-Rhizoctonia solani pathsystem by investigating H(2)O(2) burst, phenolics accumulation and analyzing the expression of phenylalanine ammonia-lyase (PAL) and peroxidase (cprx1) genes. Riboflavin was capable of priming plant defense responses via timely induction of H(2)O(2) production and phenolics accumulation. A correlation was found between induction of resistance by riboflavin and upregulation of PAL and cprx1 which are involved in phenylpropanoid signaling and phenolics metabolism. Application of peroxidase and PAL inhibitors suppressed not only basal resistance, but also riboflavin-IR of sugar beet to the pathogen. Treatment of the leaves with each inhibitor alone or together with riboflavin reduced phenolics accumulation which was correlated with higher level of disease progress. Together, these results demonstrate the indispensability of rapid H(2)O(2) accumulation, phenylpropanoid pathway and phenolics metabolism in basal defense and riboflavin-IR of sugar beet against R. solani.


Assuntos
Beta vulgaris/efeitos dos fármacos , Fármacos Fotossensibilizantes/farmacologia , Imunidade Vegetal/efeitos dos fármacos , Rhizoctonia/imunologia , Riboflavina/farmacologia , Beta vulgaris/imunologia , Beta vulgaris/microbiologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Peróxido de Hidrogênio/análise , Peróxido de Hidrogênio/metabolismo , Peroxidases/antagonistas & inibidores , Peroxidases/genética , Fenóis/metabolismo , Fenilalanina Amônia-Liase/antagonistas & inibidores , Fenilalanina Amônia-Liase/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/fisiologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/microbiologia , Folhas de Planta/fisiologia , Explosão Respiratória/efeitos dos fármacos , Rhizoctonia/patogenicidade , Transdução de Sinais , Fatores de Tempo
12.
Appl Biochem Biotechnol ; 160(7): 1933-46, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19593671

RESUMO

The chit1 gene from the entomopathogenic fungus Metarhizium anisopliae, encoding the endochitinase CHIT42, was placed under the control of the CaMV 35S promoter, and the resulting construct was transferred to tobacco. Seventeen kanamycin-resistant transgenic lines were recovered, and the presence of the transgene was confirmed by polymerase chain reactions and Southern blot hybridization. The number of chit1 copies was determined to be varying from one to four. Copy number had observable effects neither on plant growth nor development. Substantial heterogeneity concerning production of the recombinant chitinase, and both general and specific chitinolytic activities were detected in leaf extracts from primary transformants. The highest chitinase activities were found in plants harboring two copies of chit1 inserts at different loci. Progeny derived from self-pollination of the primary transgenics revealed a stable inheritance pattern, with transgene segregation following a mendelian dihybrid ratio. Two selected plants expressing high levels of CHIT42 were consistently resistant to the soilborne pathogen Rhizoctonia solani, suggesting a direct relationship between enzyme activity and reduction of foliar area affected by fungal lesions. To date, this is the first report of resistance to fungal attack in plants mediated by a recombinant chitinase from an entomopathogenic and acaricide fungus.


Assuntos
Quitinases/genética , Quitinases/metabolismo , Metarhizium/enzimologia , Nicotiana/microbiologia , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Rhizoctonia/fisiologia , Southern Blotting , Ativação Enzimática/genética , Dosagem de Genes , Perfilação da Expressão Gênica , Metarhizium/genética , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Folhas de Planta/genética , Folhas de Planta/imunologia , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Rhizoctonia/imunologia , Rhizoctonia/patogenicidade , Nicotiana/genética , Nicotiana/imunologia , Transgenes
13.
Can J Microbiol ; 51(4): 345-53, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15980897

RESUMO

Rhizoctonia solani causes yield losses in numerous economically important European crops. To develop a biocontrol strategy, 3 potato-associated ecto- and endophytically living bacterial strains Pseudomonas fluorescens B1, Pseudomonas fluorescens B2, and Serratia plymuthica B4 were evaluated against R. solani in potato and in lettuce. The disease-suppression effect of the 3 biocontrol agents (BCAs) was tested in a growth chamber and in the field. In growth chamber experiments, all 3 BCAs completely or significantly limited the dry mass (DM) losses on lettuce and the disease severity (DS) caused by R. solani on potato sprouts. Strain B1 showed the highest suppression effect (52% on average) on potato. Under field conditions, the DS on both crops, which were bacterized, decreased significantly, and the biomass losses on lettuce decreased significantly as well. The greatest disease-suppression effect on potato was achieved by strain B1 (37%), followed by B2 (33%) and then B4 (31%), whereas the marketable tuber yield increased up to 12% (B1), 6% (B2), and 17% (B4) compared with the pathogen control at higher disease pressure. Furthermore, in all experiments, B1 proved to be the most effective BCA against R. solani. Therefore, this BCA could be a candidate for developing a commercial product against Rhizoctonia diseases. To our knowledge, this is the first report on the high potential of endophytes to be used as a biological control agent against R. solani under field conditions.


Assuntos
Antibiose , Lactuca/microbiologia , Doenças das Plantas/microbiologia , Pseudomonas fluorescens/imunologia , Rhizoctonia/imunologia , Serratia/fisiologia , Solanum tuberosum/microbiologia , Agricultura/métodos , Lactuca/crescimento & desenvolvimento , Solanum tuberosum/crescimento & desenvolvimento
14.
Environ Microbiol ; 6(4): 323-34, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15008811

RESUMO

Studies of the saprotrophic growth dynamics of Trichoderma species and their fungal hosts during antagonistic interactions are severely hampered by the absence of methods that allow the unambiguous identification and quantification of individual genera in complex environments such as soil or compost containing mixed populations of fungi. Furthermore, methods are required that allow discrimination between active hyphal growth and other components of fungal biomass such as quiescent spores that are produced in large numbers by Trichoderma species. This study details the use of monoclonal antibodies to quantify the saprotrophic growth dynamics of the soil-borne plant pathogen Rhizoctonia solani and biological control strains of Trichoderma asperellum and Trichoderma harzianum during antagonistic interactions in peat-based microcosms. Quantification was based on the immunological detection of constitutive, extracellular antigens that are secreted from the growing tip of Rhizoctonia and Trichoderma mycelium and, in the case of Trichoderma harzianum, from quiescent phialoconidia also. The Trichoderma-specific monoclonal antibody (MF2) binds to a protein epitope of the enzyme glucoamylase, which was shown by immunofluorescence and immunogold electron gold microscopy studies of Trichoderma virens in vitro to be produced at the origin of germ tube emergence in phialoconidia and from the growing tip of germ tubes. In addition, a non-destructive immunoblotting technique showed that the enzyme was secreted during active growth of Trichoderma asperellum mycelium in peat. The Rhizoctonia solani-specific monoclonal antibody (EH2) similarly binds to a protein epitope of a glycoprotein that is secreted during active mycelial growth. Extracts derived from lyophilized mycelium were used as a quantifiable and repeatable source of antigens for construction of calibration curves. These curves were used to convert the absorbance values obtained in ELISA tests of peat extracts to biomass equivalents, which allowed comparisons of the saprotrophic growth dynamics of the pathogen and antagonists to be made in single or mixed species microcosms. Trichoderma species were able to compete successfully with R. solani for nutrients and to prevent saprotrophic growth of the pathogen. Specificity of the Trichoderma quantitative assay was tested in non-sterile soil-based microcosms artificially inoculated with T. asperellum. The assay was highly specific and only detected T. asperellum population dynamics. No cross-reactivity was found with extracts from soil samples containing contaminant fungi.


Assuntos
Rhizoctonia/imunologia , Rhizoctonia/fisiologia , Trichoderma/imunologia , Trichoderma/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Antígenos de Fungos/imunologia , Antígenos de Fungos/metabolismo , Ensaio de Imunoadsorção Enzimática , Glucana 1,4-alfa-Glucosidase/metabolismo , Imuno-Histoquímica , Micélio/química , Micélio/metabolismo , Rhizoctonia/citologia , Rhizoctonia/metabolismo , Solo , Microbiologia do Solo , Trichoderma/crescimento & desenvolvimento , Trichoderma/ultraestrutura
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