RESUMO
Black spot caused by Alternaria brassicicola is an important fungal disease affecting cruciferous crops, including Korean cabbage (Brassica rapa subsp. pekinensis). The interaction between Arabidopsis thaliana and Alt. brassicicola is a representative model system, and objective estimation of disease progression is indispensable for accurate functional analyses. Five strains caused black spot symptom progression on Korean cabbage and Ara. thaliana ecotype Col-0. In particular, challenge with the strains Ab44877 and Ab44414 induced severe black spot progression on Korean cabbage. Ab44877 was also highly infective on Col-0; however, the virulence of Ab44414 and the remaining strains on Col-0 was lower. To unveil the relationship between mycelial growth in the infected tissues and symptom progression, we have established a reliable quantification method using real-time PCR that employs a primer pair and dual-labelled probe specific to a unigene encoding A. brassicicola SCYTALONE DEHYDRATASE1 (AbSCD1), which is involved in fungal melanin biosynthesis. Plotting the crossing point values from the infected tissue DNA on a standard curve revealed active fungal ramification of Ab44877 in both host species. In contrast, the proliferation rate of Ab44414 in Korean cabbage was 3.8 times lower than that of Ab44877. Massive infective mycelial growth of Ab44877 was evident in Col-0; however, inoculation with Ab44414 triggered epiphytic growth rather than actual in planta ramification. Mycelial growth did not always coincide with symptom development. Our quantitative evaluation system is applicable and reliable for the objective estimation of black spot disease severity.
Assuntos
Alternaria/crescimento & desenvolvimento , Arabidopsis/microbiologia , Brassica rapa/microbiologia , Doenças das Plantas/microbiologia , Alternaria/classificação , Alternaria/genética , Alternaria/patogenicidade , Proteínas Fúngicas/genética , Micélio/classificação , Micélio/crescimento & desenvolvimento , Micélio/patogenicidade , VirulênciaRESUMO
In eukaryotes, the cell cycle consists of four distinct phases: G1, S, G2 and M. In certain condition, the cells skip M-phase and undergo endoreduplication. Endoreduplication, occurring during a modified cell cycle, duplicates the entire genome without being followed by M-phase. A cycle of endoreduplication is common in most of the differentiated cells of plant vegetative tissues and it occurs extensively in cereal endosperm cells. Endoreduplication occurs when CDK/Cyclin complex low or inactive caused by ubiquitin-mediated degradation by APC and their activators. In this study, rice cell cycle switch 52 A (OsCCS52A), an APC activator, is functionally characterized using the reverse genetic approach. In rice, OsCCS52A is highly expressed in seedlings, flowers, immature panicles and 15 DAP kernels. Localization studies revealed that OsCCS52A is a nuclear protein. OsCCS52A interacts with OsCdc16 in yeast. In addition, overexpression of OsCCS52A inhibits mitotic cell division and induces endoreduplication and cell elongation in fission yeast. The homozygous mutant exhibits dwarfism and smaller seeds. Further analysis demonstrated that endoreduplication cycles in the endosperm of mutant seeds were disturbed, evidenced by reduced nuclear and cell sizes. Taken together, these results suggest that OsCCS52A is involved in maintaining normal seed size formation by mediating the exit from mitotic cell division to enter the endoreduplication cycles in rice endosperm.
Assuntos
Endosperma/genética , Oryza/genética , Proteínas de Plantas/metabolismo , RNA de Plantas/genética , Sequência de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Crescimento Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Tamanho Celular , Clonagem Molecular , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Produtos Agrícolas/metabolismo , Endosperma/crescimento & desenvolvimento , Endosperma/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Mitose , Dados de Sequência Molecular , Mutação , Fases de Leitura Aberta , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Componentes Aéreos da Planta/genética , Componentes Aéreos da Planta/crescimento & desenvolvimento , Componentes Aéreos da Planta/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Polinização , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Transformação Genética , Técnicas do Sistema de Duplo-Híbrido , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
⢠This study reports that Arabidopsis thaliana protein serine/threonine phosphatase 5 (AtPP5) plays a pivotal role in heat stress resistance. A high-molecular-weight (HMW) form of AtPP5 was isolated from heat-treated A. thaliana suspension cells. AtPP5 performs multiple functions, acting as a protein phosphatase, foldase chaperone, and holdase chaperone. The enzymatic activities of this versatile protein are closely associated with its oligomeric status, ranging from low oligomeric protein species to HMW complexes. ⢠The phosphatase and foldase chaperone functions of AtPP5 are associated primarily with the low-molecular-weight (LMW) form, whereas the HMW form exhibits holdase chaperone activity. Transgenic over-expression of AtPP5 conferred enhanced heat shock resistance to wild-type A. thaliana and a T-DNA insertion knock-out mutant was defective in acquired thermotolerance. A recombinant phosphatase mutant (H290N) showed markedly increased holdase chaperone activity. ⢠In addition, enhanced thermotolerance was observed in transgenic plants over-expressing H290N, which suggests that the holdase chaperone activity of AtPP5 is primarily responsible for AtPP5-mediated thermotolerance. ⢠Collectively, the results from this study provide the first evidence that AtPP5 performs multiple enzymatic activities that are mediated by conformational changes induced by heat-shock stress.
Assuntos
Arabidopsis/fisiologia , Resposta ao Choque Térmico/fisiologia , Chaperonas Moleculares/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Adaptação Fisiológica , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Arabidopsis/metabolismo , Células Cultivadas , Regulação da Expressão Gênica de Plantas , Biblioteca Gênica , Temperatura Alta , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/isolamento & purificação , Proteínas Nucleares/metabolismo , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/isolamento & purificação , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Multimerização Proteica , Proteínas RecombinantesRESUMO
p23 is a heat shock protein 90 (Hsp90) co-chaperone and stabilizes the Hsp90 heterocomplex in mammals and yeast. In this study, we isolated a complementary DNA (cDNA) encoding p23 from orchardgrass (Dgp23) and characterized its functional roles under conditions of thermal stress. Dgp23 is a 911 bp cDNA with an open reading frame predicted to encode a 180 amino acid protein. Northern analysis showed that expression of Dgp23 transcripts was heat inducible. Dgp23 has a well-conserved p23 domain and interacted with an orchardgrass Hsp90 homolog in vivo, like mammalian and yeast p23 homologs. Recombinant Dgp23 is a small acidic protein with a molecular mass of approximately 27 kDa and pI 4.3. Dgp23 was also shown to function as a chaperone protein by suppression of malate dehydrogenase thermal aggregation. Differential scanning calorimetry thermograms indicated that Dgp23 is a heat-stable protein, capable of increasing the T (m) of lysozyme. Moreover, overexpression of Dgp23 in a yeast p23 homolog deletion strain, Deltasba1, increased cell viability. These results suggest that Dgp23 plays a role in thermal stress-tolerance and functions as a co-chaperone of Hsp90 and as a chaperone.
Assuntos
Dactylis/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Animais , Dactylis/genética , Teste de Complementação Genética , Proteínas de Choque Térmico HSP90/genética , Temperatura Alta , Humanos , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Fases de Leitura Aberta , Proteínas de Plantas/genética , Desnaturação Proteica , Alinhamento de Sequência , Técnicas do Sistema de Duplo-HíbridoRESUMO
Rhizoctonia solani has a wide host range, including almost all cultivated crops and its subgroup anastomosis group (AG)-1 IA causes sheath blight in rice. An accurate measurement of pathogen's biomass is a convincing tool for enumeration of this disease. Mycological characteristics and molecular diagnosis simultaneously supported that all six strains in this study were R. solani AG-1 IA. Heterokaryons between strains Rs40104, Rs40105, and Rs45811 were stable and viable, whereas Rs40103 and Rs40106 did not form viable fused cells, except for the combination of Rs40106 and Rs40104. A primer pair was highly specific to RsAROM gene of R. solani strains and the amplified fragment exists as double copies within fungal genome. The relationship between crossing point (CP) values and the amount of fungal DNA was reliable (R (2) >0.99). Based on these results, we determined R. solani's proliferation within infected stems through real time PCR using a primer pair and a Taqman probe specific to the RsAROM gene. The amount of fungal DNA within the 250 ng of tissue DNA from rice cv. Dongjin infected with Rs40104, Rs40105, and Rs45811 were 7.436, 5.830, and 5.085 ng, respectively. In contrast, the fungal DNAs within the stems inoculated with Rs40103 and Rs40106 were 0.091 and 0.842 ng. The sheath blight symptom progression approximately coincided with the amount of fungal DNA within the symptoms. In summary, our quantitative evaluation method provided reliable and objective results reflecting the amount of fungal biomass within the infected tissues and would be useful for evaluation of resistance germplasm or fungicides and estimation of inoculum potential.
Assuntos
Oryza/microbiologia , Rhizoctonia/genética , DNA Fúngico/genética , Genoma Fúngico/genética , Doenças das Plantas/microbiologia , Reação em Cadeia da Polimerase em Tempo Real , Rhizoctonia/patogenicidadeRESUMO
The seven members of the 90-kDa heat shock protein (Hsp90) family encode highly conserved molecular chaperones essential for cell survival in Arabidopsis thaliana. Hsp90 are abundant proteins, localized in different compartments with AtHsp90.1-4 in the cytosol and AtHsp90.5-7 in different organelles. Among the AtHsp90, AtHsp90.1, is stress-inducible and shares comparatively low sequence identity with the constitutively expressed AtHsp90.2-4. Even though abundant information is available on mammalian cytosolic Hsp90 proteins, it is unknown whether cytosolic Hsp90 proteins display different structural and functional properties. We have now analyzed two A. thalianas cytosolic Hsp90s, AtHsp90.1 and AtHsp90.3, for functional divergence. AtHsp90.3 showed higher holdase chaperone activity than AtHsp90.1, although both AtHsp90s exhibited effective chaperone activity. Size-exclusion chromatography revealed different oligomeric states distinguishing the two Hsp90 proteins. While AtHsp90.1 exists in several oligomeric states, including monomers, dimers and higher oligomers, AtHsp90.3 exists predominantly in a high oligomeric state. High oligomeric state of AtHsp90.1 showed higher holdase chaperone activity than the respective monomer or dimer states. When high oligomeric forms of AtHsp90.1 and AtHsp90.3 are reduced by DTT, activity was reduced compared to that found in the native high oligomeric state. In addition, ATP-dependent foldase chaperone activity of AtHsp90.3 was higher with strong intrinsic ATPase activity than that of AtHsp90.1. As a conclusion, the two A. thaliana cytosolic Hsp90 proteins display different functional activities depending on structural differences, implying functional divergence although the proteins are localized to the same sub-cellular organelle.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Citosol/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Trifosfato de Adenosina/metabolismo , DimerizaçãoRESUMO
Rice blast caused by Magnaporthe oryzae is a major disease in the paddy field and also a representative model system in the investigation of plant-microbe interactions. This study was undertaken to provide the quantitative evaluation method that specifically determines the amount of M. oryzae proliferation in planta. Real-time PCR was used as the detection strategy in combination with the primer pair and Taqman probe specific to MHP1, a unigene encoding HYDROPHOBIN that is indispensable for normal virulence expression. Based on the crossing point values from the PCR reactions containing a series of increasing concentration of cloned amplicon or fungal genomic DNA, correlation among the template's copy number or its amount and amplification pattern was calculated. Reliability of this equation was further confirmed using the DNA samples from the rice leaves infected with compatible or incompatible strains of M. oryzae. The primer pair used in the Taqman real-time PCR reaction can recognize the existence of fungal DNA as low as 1 pg. In sum, our quantitative evaluation system is applicable and reliable in the blast diagnosis and also in the estimation of objective blast disease progression.
Assuntos
DNA Fúngico/análise , Proteínas Fúngicas/análise , Magnaporthe/crescimento & desenvolvimento , Oryza/microbiologia , Doenças das Plantas/microbiologia , Folhas de Planta/microbiologia , Fatores de Virulência/análise , Primers do DNA , DNA Fúngico/genética , Eletroforese em Gel de Ágar , Proteínas Fúngicas/biossíntese , Proteínas Fúngicas/genética , Dosagem de Genes , Magnaporthe/patogenicidade , Reação em Cadeia da Polimerase em Tempo Real , Fatores de Tempo , Fatores de Virulência/biossíntese , Fatores de Virulência/genéticaRESUMO
Rice brown leaf spot is a major disease in the rice paddy field. The causal agent Cochliobolus miyabeanus is an ascomycete fungus and a representative necrotrophic pathogen in the investigation of rice-microbe interactions. The aims of this research were to identify a quantitative evaluation method to determine the amount of C. miyabeanus proliferation in planta and determine the method's sensitivity. Real-time polymerase chain reaction (PCR) was employed in combination with the primer pair and Taqman probe specific to CmSCD1, a C. miyabeanus unigene encoding SCYTALONE DEHYDRATASE, which is involved in fungal melanin biosynthesis. Comparative analysis of the nucleotide sequences of CmSCD1 from Korean strains with those from the Japanese and Taiwanese strains revealed some sequence differences. Based on the crossing point (CP) values from Taqman real-time PCR containing a series of increasing concentrations of cloned amplicon or fungal genomic DNA, linear regressions with a high level of reliability (R(2)>0.997) were constructed. This system was able to estimate fungal genomic DNA at the picogram level. The reliability of this equation was further confirmed using DNA samples from both resistant and susceptible cultivars infected with C. miyabeanus. In summary, our quantitative system is a powerful alternative in brown leaf spot forecasting and in the consistent evaluation of disease progression.
Assuntos
Ascomicetos/genética , Ascomicetos/metabolismo , Hidroliases/genética , Melaninas/biossíntese , Oryza/microbiologia , Doenças das Plantas/microbiologia , Sequência de Aminoácidos , Sequência de Bases , Evolução Biológica , DNA Fúngico/genética , Hidroliases/química , Hidroliases/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase em Tempo Real , Alinhamento de SequênciaRESUMO
When plants are exposed to extreme temperature, stress-inducible proteins are highly induced and involved in subcellular defence mechanisms. Hsp70, one of stress-inducible proteins, functions as an ATP-dependent molecular chaperone in broad organisms to process such as the inhibition of protein denaturation, promotion of protein folding, and renaturation of denatured proteins. In this study, we isolated a heat-inducible orchardgrass Hsp70 (DgHsp70) that is a homolog of cytosolic Hsp70 that possesses a CaM-binding domain. Purified DgHsp70 protein displayed dose-dependent ATPase, holdase, and ATP-dependent foldase activities. To investigate functional roles of DgHsp70 by the association of Arabidopsis calmodulin-2 (AtCaM2), showing heat-sensitive reduction on transcription, we first characterized the binding activity by gel-overlay assay. DgHsp70 binds to AtCaM2 in the presence of Ca(2+) via a conserved CaM-binding domain. Ca(2+)/AtCaM2 binding decreased ATPase activity of DgHsp70, and concomitantly, reduced foldase activity. Based on the protein structure of bovine Hsc70, which is the closest structural homolog of DgHsp70, a CaM-binding domain is located near the ATP-binding site and CaM may span the ATP-binding pocket of Hsp70. Its decreased functional foldase activity may be caused by blocking ATP hydrolysis after Ca(2+)/AtCaM2 binding. It may associate with inhibition of functional activity of DgHsp70 in the absence of stress and/or de novo protein synthesis of DgHsp70 in the presence of thermal stress condition.
Assuntos
Adaptação Fisiológica , Arabidopsis/metabolismo , Cálcio/metabolismo , Calmodulina/metabolismo , Dactylis/química , Proteínas de Choque Térmico HSP70/metabolismo , Temperatura Alta , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação , Bovinos , Proteínas de Choque Térmico HSP70/química , Hidrólise , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Ligação Proteica , Estresse FisiológicoRESUMO
Two strains of necrotrophic Alternaria brassicicola, Ab40857 and Ab42464, are virulent on Korean cabbage and several wild types of Arabidopsis thaliana. Interaction between Ab42464 and Col-0 was compatible, whereas interaction between Ab40857 and Col-0 was incompatible. The loss of defense, no death (dnd) 1 function abrogated the compatibility between Ab42464 and Col-0, and the accelerated cell death (acd) 2 mutation attenuated the Col-0's resistance against Ab40857. These two fungal strains induced PR1 transcription in Col-0. Ab40857 accelerated transcription of PDF1.2, THI2.1, CAT, and POX by 12 h compared to those challenged with Ab42464. More abundant cell death was observed in Col-0 infected with Ab42464, however, callose deposition was evident in the incompatible interaction. Remarkably, Ab40857-infected areas of acd2-2 underwent rampant cell death and Ab42464 triggered callose production in dnd1-1. Furthermore, the incompatibility between Ab40857 and Col-0 was nullified by the coronatine-insensitive 1 (coi1) and phytoalexin-deficient 3 (pad3) mutations but not by nonexpresser of PR genes (npr1) and pad4. Ab40857 induced abundant cell death in pad3. Taken together, cell death during the early infection stage is a key determinant that discriminates between a compatible interaction and an incompatible one, and the resistance within Col-0 against Ab40857 is dependent on a defense-signaling pathway mediated by jasmonic acid and PAD3.
Assuntos
Alternaria/fisiologia , Arabidopsis/microbiologia , Interações Hospedeiro-Patógeno , Alternaria/patogenicidade , Arabidopsis/citologia , Arabidopsis/genética , Morte Celular/genética , Perfilação da Expressão Gênica , Peróxido de Hidrogênio/metabolismo , Indóis/metabolismo , Doenças das Plantas/microbiologia , Imunidade Vegetal , Folhas de Planta/citologia , Folhas de Planta/genética , Folhas de Planta/microbiologia , Tiazóis/metabolismoRESUMO
Hsp90 proteins are essential molecular chaperones regulating multiple cellular processes in distinct subcellular organelles. In this study, we report the functional characterization of a cDNA encoding endoplasmic reticulum (ER)-resident Hsp90 from orchardgrass (DgHsp90). DgHsp90 is a 2742bp cDNA with an open reading frame predicted to encode an 808 amino acid protein. DgHsp90 has a well conserved N-terminal ATPase domain and a C-terminal Hsp90 domain and ER-retention motif. Expression of DgHsp90 increased during heat stress at 35 degrees C or H(2)O(2) treatment. DgHsp90 also functions as a chaperone protein by preventing thermal aggregation of malate dehydrogenase (EC 1.1.1.37) and citrate synthase (EC 2.3.3.1). The intrinsic ATPase activity of DgHsp90 was inhibited by geldanamycin, an Hsp90 inhibitor, and the inhibition reduced the chaperone activity of DgHsp90. Yeast cells overexpressing DgHsp90 exhibited enhanced thermotolerance.
Assuntos
Adenosina Trifosfatases/metabolismo , Dactylis/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas de Plantas/metabolismo , Adaptação Fisiológica , Adenosina Trifosfatases/genética , Sequência de Aminoácidos , Sequência de Bases , Benzoquinonas/farmacologia , Northern Blotting , DNA Complementar/química , DNA Complementar/genética , Dactylis/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas de Choque Térmico HSP90/classificação , Proteínas de Choque Térmico HSP90/genética , Temperatura Alta , Peróxido de Hidrogênio/farmacologia , Lactamas Macrocíclicas/farmacologia , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Oxidantes/farmacologia , Filogenia , Proteínas de Plantas/classificação , Proteínas de Plantas/genética , Análise de Sequência de DNARESUMO
Increasing its root to shoot ratio is a plant strategy for restoring water homeostasis in response to the long-term imposition of mild water stress. In addition to its important role in diverse fundamental processes, indole-3-acetic acid (IAA) is involved in root growth and development. Recent extensive characterizations of the YUCCA gene family in Arabidopsis and rice have elucidated that member's function in a tryptophan-dependent IAA biosynthetic pathway. Through forward- and reverse-genetics screening, we have isolated Tos17 and T-DNA insertional rice mutants in a CONSTITUTIVELY WILTED1 (COW1) gene, which encodes a new member of the YUCCA protein family. Homozygous plants with either a Tos17 or T-DNA-inserted allele of OsCOW1 exhibit phenotypes of rolled leaves, reduced leaf widths, and lower root to shoot ratios. These phenotypes are evident in seedlings as early as 7-10 d after germination, and remain until maturity. When oscow1 seedlings are grown under low-intensity light and high relative humidity, the rolled-leaf phenotype is greatly alleviated. For comparison, in such conditions, the transpiration rate for WT leaves decreases approx. 5- to 10-fold, implying that this mutant trait results from wilting rather than being a morphogenic defect. Furthermore, a lower turgor potential and transpiration rate in their mature leaves indicates that oscow1 plants are water-deficient, due to insufficient water uptake that possibly stems from that diminished root to shoot ratio. Thus, our observations suggest that OsCOW1-mediated IAA biosynthesis plays an important role in maintaining root to shoot ratios and, in turn, affects water homeostasis in rice.