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1.
Int J Mol Sci ; 22(19)2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34639149

RESUMO

Fungal enzymes degrading the plant cell wall, such as xylanases, can activate plant immune responses. The Fusarium graminearum FGSG_03624 xylanase, previously shown to elicit necrosis and hydrogen peroxide accumulation in wheat, was investigated for its ability to induce disease resistance. To this aim, we transiently and constitutively expressed an enzymatically inactive form of FGSG_03624 in tobacco and Arabidopsis, respectively. The plants were challenged with Pseudomonas syringae pv. tabaci or pv. maculicola and Botrytis cinerea. Symptom reduction by the bacterium was evident, while no reduction was observed after B. cinerea inoculation. Compared to the control, the presence of the xylanase gene in transgenic Arabidopsis plants did not alter the basal expression of a set of defense-related genes, and, after the P. syringae inoculation, a prolonged PR1 expression was detected. F. graminearum inoculation experiments of durum wheat spikes exogenously treated with the FGSG_03624 xylanase highlighted a reduction of symptoms in the early phases of infection and a lower fungal biomass accumulation than in the control. Besides, callose deposition was detected in infected spikes previously treated with the xylanase and not in infected control plants. In conclusion, our results highlight the ability of FGSG_03624 to enhance plant immunity, thus decreasing disease severity.


Assuntos
Arabidopsis/imunologia , Botrytis/patogenicidade , Resistência à Doença/imunologia , Endo-1,4-beta-Xilanases/metabolismo , Fusarium/enzimologia , Imunidade Vegetal , Pseudomonas syringae/patogenicidade , Tabaco/imunologia , Arabidopsis/metabolismo , Arabidopsis/microbiologia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Tabaco/metabolismo , Tabaco/microbiologia
2.
Nat Commun ; 12(1): 5969, 2021 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-34645811

RESUMO

The Yersinia outer protein J (YopJ) family effectors are widely deployed through the type III secretion system by both plant and animal pathogens. As non-canonical acetyltransferases, the enzymatic activities of YopJ family effectors are allosterically activated by the eukaryote-specific ligand inositol hexaphosphate (InsP6). However, the underpinning molecular mechanism remains undefined. Here we present the crystal structure of apo-PopP2, a YopJ family member secreted by the plant pathogen Ralstonia solanacearum. Structural comparison of apo-PopP2 with the InsP6-bound PopP2 reveals a substantial conformational readjustment centered in the substrate-binding site. Combining biochemical and computational analyses, we further identify a mechanism by which the association of InsP6 with PopP2 induces an α-helix-to-ß-strand transition in the catalytic core, resulting in stabilization of the substrate recognition helix in the target protein binding site. Together, our study uncovers the molecular basis governing InsP6-mediated allosteric regulation of YopJ family acetyltransferases and further expands the paradigm of fold-switching proteins.


Assuntos
Acetiltransferases/química , Apoproteínas/química , Arabidopsis/microbiologia , Proteínas de Bactérias/química , Ácido Fítico/química , Ralstonia solanacearum/química , Acetiltransferases/genética , Acetiltransferases/metabolismo , Regulação Alostérica , Apoproteínas/genética , Apoproteínas/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Ácido Fítico/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Ralstonia solanacearum/enzimologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade por Substrato , Tabaco/microbiologia
3.
BMC Plant Biol ; 21(1): 425, 2021 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-34537002

RESUMO

BACKGROUND: The Catharanthus roseus RLK1-like kinase (CrRLK1L) is a subfamily of the RLK gene family, and members are sensors of cell wall integrity and regulators of cell polarity growth. Recent studies have also shown that members of this subfamily are involved in plant immunity. Nicotiana benthamiana is a model plant widely used in the study of plant-pathogen interactions. However, the members of the NbCrRLK1L subfamily and their response to pathogens have not been reported. RESULTS: In this study, a total of 31 CrRLK1L members were identified in the N. benthamiana genome, and these can be divided into 6 phylogenetic groups (I-VI). The members in each group have similar exon-intron structures and conserved motifs. NbCrRLK1Ls were predicted to be regulated by cis-acting elements such as STRE, TCA, ABRE, etc., and to be the target of transcription factors such as Dof and MYB. The expression profiles of the 16 selected NbCrRLK1Ls were determined by quantitative PCR. Most NbCrRLK1Ls were highly expressed in leaves but there were different and diverse expression patterns in other tissues. Inoculation with the bacterium Pseudomonas syringae or with Turnip mosaic virus significantly altered the transcript levels of the tested genes, suggesting that NbCrRLK1Ls may be involved in the response to pathogens. CONCLUSIONS: This study systematically identified the CrRLK1L members in N. benthamiana, and analyzed their tissue-specific expression and gene expression profiles in response to different pathogens and two pathogens associated molecular patterns (PAMPs). This research lays the foundation for exploring the function of NbCrRLK1Ls in plant-microbe interactions.


Assuntos
Catharanthus/genética , Proteínas de Plantas/genética , Proteínas Quinases/genética , Tabaco/genética , Catharanthus/enzimologia , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Interações Hospedeiro-Patógeno , Filogenia , Imunidade Vegetal/genética , Folhas de Planta/genética , Folhas de Planta/virologia , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Domínios Proteicos , Proteínas Quinases/metabolismo , Pseudomonas syringae/patogenicidade , Tabaco/microbiologia , Tabaco/virologia , Fatores de Transcrição/genética
4.
Sci Rep ; 11(1): 16360, 2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34381095

RESUMO

Tobacco bacterial wilt (TBW) is seriously damages the growth of tobacco. There is an urgent need to find a safer and more effective measure to control TBW. In this study, B. amyloliquefaciens ZM9 and marigold powder were applied to the tobacco roots alone or in combination, and the potential inhibition of TBW was assessed. On the other hand, the effects of these treatments on soil physicochemical properties, rhizosphere microbial community and soil metabolites were also evaluated. The results showed that the application of B. amyloliquefaciens ZM9 or marigold powder alone significantly reduced the abundance of R. solanacearum in rhizosphere soil, while the integrated treatment showed the strongest inhibitory effect. Moreover, the integrated treatment can inhibit the secretion of chemoattractants, and affect the change of rhizosphere soil microbial composition. In conclusion, the combination of antagonistic bacteria agent B. amyloliquefaciens ZM9 with marigold powder can enhance the suppression of TBW. Furthermore, B. amyloliquefaciens ZM9 and marigold have synergistic effects on suppressing TBW by regulation soil physicochemical properties, soil metabolites and microbial structure. This study provide a promising strategy for TBW control by integrated applying of B. amyloliquefaciens ZM9 and marigold powder.


Assuntos
Bacillus/fisiologia , Agentes de Controle Biológico/administração & dosagem , Microbiota/fisiologia , Doenças das Plantas/microbiologia , Pós/administração & dosagem , Tabaco/microbiologia , Raízes de Plantas/microbiologia , Rizosfera , Solo , Microbiologia do Solo
5.
PLoS Biol ; 19(7): e3001326, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34260583

RESUMO

Arbuscular mycorrhiza (AM) are mutualistic interactions formed between soil fungi and plant roots. AM symbiosis is a fundamental and widespread trait in plants with the potential to sustainably enhance future crop yields. However, improving AM fungal association in crop species requires a fundamental understanding of host colonisation dynamics across varying agronomic and ecological contexts. To this end, we demonstrate the use of betalain pigments as in vivo visual markers for the occurrence and distribution of AM fungal colonisation by Rhizophagus irregularis in Medicago truncatula and Nicotiana benthamiana roots. Using established and novel AM-responsive promoters, we assembled multigene reporter constructs that enable the AM-controlled expression of the core betalain synthesis genes. We show that betalain colouration is specifically induced in root tissues and cells where fungal colonisation has occurred. In a rhizotron setup, we also demonstrate that betalain staining allows for the noninvasive tracing of fungal colonisation along the root system over time. We present MycoRed, a useful innovative method that will expand and complement currently used fungal visualisation techniques to advance knowledge in the field of AM symbiosis.


Assuntos
Betalaínas/metabolismo , Micorrizas/crescimento & desenvolvimento , Genes Fúngicos , Marcadores Genéticos , Medicago truncatula/microbiologia , Micorrizas/genética , Micorrizas/metabolismo , Raízes de Plantas/microbiologia , Regiões Promotoras Genéticas , Simbiose/genética , Tabaco/genética , Tabaco/microbiologia
6.
Sci Rep ; 11(1): 12182, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34108579

RESUMO

Stress caused by pathogens strongly damages plants. Developing products to control plant disease is an important challenge in sustainable agriculture. In this study, a heat-killed endophytic bacterium (HKEB), Bacillus aryabhattai, is used to induce plant defense against fungal and bacterial pathogens, and the main defense pathways used by the HKEB to activate plant defense are revealed. The HKEB induced high protection against different pathogens through the salicylic and jasmonic acid pathways. We report the presence of gentisic acid in the HKEB for the first time. These results show that HKEBs may be a useful tool for the management of plant diseases.


Assuntos
Arabidopsis/metabolismo , Bacillus/fisiologia , Gentisatos/metabolismo , Temperatura Alta , Doenças das Plantas/imunologia , Proteínas de Plantas/metabolismo , Tabaco/metabolismo , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/microbiologia , Bacillus/química , Ciclopentanos/metabolismo , Regulação da Expressão Gênica de Plantas , Oxilipinas/metabolismo , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Ácido Salicílico/metabolismo , Tabaco/genética , Tabaco/imunologia , Tabaco/microbiologia
7.
J Virol ; 95(17): e0026421, 2021 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-34132570

RESUMO

Uncharacterized viral genomes that encode circular replication-associated proteins of single-stranded DNA viruses have been discovered by metagenomics/metatranscriptomics approaches. Some of these novel viruses are classified in the newly formed family Genomoviridae. Here, we determined the host range of a novel genomovirus, SlaGemV-1, through the transfection of Sclerotinia sclerotiorum with infectious clones. Inoculating with the rescued virions, we further transfected Botrytis cinerea and Monilinia fructicola, two economically important members of the family Sclerotiniaceae, and Fusarium oxysporum. SlaGemV-1 causes hypovirulence in S. sclerotiorum, B. cinerea, and M. fructicola. SlaGemV-1 also replicates in Spodoptera frugiperda insect cells but not in Caenorhabditis elegans or plants. By expressing viral genes separately through site-specific integration, the replication protein alone was sufficient to cause debilitation. Our study is the first to demonstrate the reconstruction of a metagenomically discovered genomovirus without known hosts with the potential of inducing hypovirulence, and the infectious clone allows for studying mechanisms of genomovirus-host interactions that are conserved across genera. IMPORTANCE Little is known about the exact host range of widespread genomoviruses. The genome of soybean leaf-associated gemygorvirus-1 (SlaGemV-1) was originally assembled from a metagenomic/metatranscriptomic study without known hosts. Here, we rescued SlaGemV-1 and found that it could infect three important plant-pathogenic fungi and fall armyworm (S. frugiperda Sf9) insect cells but not a model nematode, C. elegans, or model plant species. Most importantly, SlaGemV-1 shows promise for inducing hypovirulence of the tested fungal species in the family Sclerotiniaceae, including Sclerotinia sclerotiorum, Botrytis cinerea, and Monilinia fructicola. The viral determinant of hypovirulence was further identified as replication initiation protein. As a proof of concept, we demonstrate that viromes discovered in plant metagenomes can be a valuable genetic resource when novel viruses are rescued and characterized for their host range.


Assuntos
Ascomicetos/virologia , Geminiviridae/isolamento & purificação , Especificidade de Hospedeiro , Metagenoma , Doenças das Plantas/prevenção & controle , Tabaco/crescimento & desenvolvimento , Virulência , Animais , Ascomicetos/genética , Ascomicetos/patogenicidade , Botrytis/genética , Botrytis/patogenicidade , Botrytis/virologia , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/microbiologia , Caenorhabditis elegans/virologia , Fusarium/genética , Fusarium/patogenicidade , Fusarium/virologia , Geminiviridae/classificação , Geminiviridae/genética , Genoma Viral , Controle Biológico de Vetores , Doenças das Plantas/microbiologia , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/microbiologia , Folhas de Planta/virologia , Soja/crescimento & desenvolvimento , Soja/microbiologia , Tabaco/microbiologia , Tabaco/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion
8.
Int J Mol Sci ; 22(10)2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-34065885

RESUMO

Genetic engineering of plants has turned out to be an attractive approach to produce various secondary metabolites. Here, we attempted to produce kynurenine, a health-promoting metabolite, in plants of Nicotiana tabacum (tobacco) transformed by Agrobacterium tumefaciens with the gene, coding for human indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme responsible for the kynurenine production because of tryptophan degradation. The presence of IDO1 gene in transgenic plants was confirmed by PCR, but the protein failed to be detected. To confer higher stability to the heterologous human IDO1 protein and to provide a more sensitive method to detect the protein of interest, we cloned a gene construct coding for IDO1-GFP. Analysis of transiently transfected tobacco protoplasts demonstrated that the IDO1-GFP gene led to the expression of a detectable protein and to the production of kynurenine in the protoplast medium. Interestingly, the intracellular localisation of human IDO1 in plant cells is similar to that found in mammal cells, mainly in cytosol, but in early endosomes as well. To the best of our knowledge, this is the first report on the expression of human IDO1 enzyme capable of secreting kynurenines in plant cells.


Assuntos
Agrobacterium tumefaciens/fisiologia , Proteínas de Fluorescência Verde/genética , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Cinurenina/metabolismo , Tabaco/microbiologia , Agrobacterium tumefaciens/genética , Clonagem Molecular , Proteínas de Fluorescência Verde/metabolismo , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Plasmídeos/genética , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Tabaco/genética , Tabaco/metabolismo , Transformação Bacteriana
9.
Sci Rep ; 11(1): 13435, 2021 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-34183701

RESUMO

Stenotrophomonas maltophilia has plant growth-promoting potential, and interaction with Arachis hypogaea changes host-plant physiology, biochemistry, and metabolomics, which provides tolerance under the N2 starvation conditions. About 226 suppression subtractive hybridization clones were obtained from plant-microbe interaction, of which, about 62% of gene sequences were uncharacterized, whereas 23% of sequences were involved in photosynthesis. An uncharacterized SSH clone, SM409 (full-length sequence showed resemblance with Cytb6), showed about 4-fold upregulation during the interaction was transformed to tobacco for functional validation. Overexpression of the AhCytb6 gene enhanced the seed germination efficiency and plant growth under N2 deficit and salt stress conditions compared to wild-type and vector control plants. Results confirmed that transgenic lines maintained high photosynthesis and protected plants from reactive oxygen species buildup during stress conditions. Microarray-based whole-transcript expression of host plants showed that out of 272,410 genes, 8704 and 24,409 genes were significantly (p < 0.05) differentially expressed (> 2 up or down-regulated) under N2 starvation and salt stress conditions, respectively. The differentially expressed genes belonged to different regulatory pathways. Overall, results suggested that overexpression of AhCytb6 regulates the expression of various genes to enhance plant growth under N2 deficit and abiotic stress conditions by modulating plant physiology.


Assuntos
Arachis/genética , Citocromos b6/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Fixação de Nitrogênio/genética , Nitrogênio/deficiência , Proteínas de Plantas/genética , Estresse Salino/genética , Stenotrophomonas maltophilia/fisiologia , Simbiose/genética , Tabaco/genética , Arachis/enzimologia , Biomassa , Mudança Climática , Simulação por Computador , Citocromos b6/fisiologia , Modelos Genéticos , Nitrogênio/metabolismo , Fotossíntese , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Tabaco/enzimologia , Tabaco/crescimento & desenvolvimento , Tabaco/microbiologia , Regulação para Cima
10.
mBio ; 12(3): e0084621, 2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34044592

RESUMO

Plant roots constitute the primary interface between plants and soilborne microorganisms and harbor microbial communities called the root microbiota. Recent studies have demonstrated a significant contribution of plant specialized metabolites (PSMs) to the assembly of root microbiota. However, the mechanistic and evolutionary details underlying the PSM-mediated microbiota assembly and its contribution to host specificity remain elusive. Here, we show that the bacterial genus Arthrobacter is predominant specifically in the tobacco endosphere and that its enrichment in the tobacco endosphere is partially mediated by a combination of two unrelated classes of tobacco-specific PSMs, santhopine and nicotine. We isolated and sequenced Arthrobacter strains from tobacco roots as well as soils treated with these PSMs and identified genomic features, including but not limited to genes for santhopine and nicotine catabolism, that are associated with the ability to colonize tobacco roots. Phylogenomic and comparative analyses suggest that these genes were gained in multiple independent acquisition events, each of which was possibly triggered by adaptation to particular soil environments. Taken together, our findings illustrate a cooperative role of a combination of PSMs in mediating plant species-specific root bacterial microbiota assembly and suggest that the observed interaction between tobacco and Arthrobacter may be a consequence of an ecological fitting process. IMPORTANCE Host secondary metabolites have a crucial effect on the taxonomic composition of its associated microbiota. It is estimated that a single plant species produces hundreds of secondary metabolites; however, whether different classes of metabolites have distinctive or common roles in the microbiota assembly remains unclear. Here, we show that two unrelated classes of secondary metabolites in tobacco play a cooperative role in the formation of tobacco-specific compositions of the root bacterial microbiota, which has been established as a consequence of independent evolutionary events in plants and bacteria triggered by different ecological effects. Our findings illustrate mechanistic and evolutionary aspects of the microbiota assembly that are mediated by an arsenal of plant secondary metabolites.


Assuntos
Arthrobacter/genética , Arthrobacter/metabolismo , Genoma Bacteriano , Interações entre Hospedeiro e Microrganismos/genética , Raízes de Plantas/microbiologia , Tabaco/microbiologia , Endófitos/genética , Endófitos/metabolismo , Interações entre Hospedeiro e Microrganismos/fisiologia , Filogenia , Raízes de Plantas/metabolismo , RNA Ribossômico 16S/genética , Rizosfera , Metabolismo Secundário , Análise de Sequência de DNA , Microbiologia do Solo , Tabaco/metabolismo
11.
Microbiologyopen ; 10(2): e1171, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33970539

RESUMO

Cigar tobacco leaves (CTLs) contain abundant bacteria and fungi that are vital to leaf quality during fermentation. In this study, artificial fermentation was used for the fermentation of CTLs since it was more controllable and efficient than natural aging. The bacterial and fungal community structure and composition in unfermented and fermented CTLs were determined to understand the effects of microbes on the characteristics of CTLs during artificial fermentation. The relationship between the chemical contents and alterations in the microbial composition was evaluated, and the functions of bacteria and fungi in fermented CTLs were predicted to determine the possible metabolic pathways. After artificial fermentation, the bacterial and fungal community structure significantly changed in CTLs. The total nitrate and nicotine contents were most readily affected by the bacterial and fungal communities, respectively. FAPROTAX software predictions of the bacterial community revealed increases in functions related to compound transformation after fermentation. FUNGuild predictions of the fungal community revealed an increase in the content of saprotrophic fungi after fermentation. These data provide information regarding the artificial fermentation mechanism of CTLs and will inform safety and quality improvements.


Assuntos
Bactérias/metabolismo , Fungos/metabolismo , Microbiota , Folhas de Planta/microbiologia , Tabaco/microbiologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Qualidade de Produtos para o Consumidor , Fermentação , Fungos/classificação , Fungos/genética , Fungos/isolamento & purificação , Humanos , Produtos do Tabaco/microbiologia
12.
Mol Plant Pathol ; 22(6): 710-726, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33835616

RESUMO

Fus3/Kss1, also known as Pmk1 in several pathogenic fungi, is a component of the mitogen-activated protein kinase (MAPK) signalling pathway that functions as a regulator in fungal development, stress response, mating, and pathogenicity. Cytospora chrysosperma, a notorious woody plant-pathogenic fungus, causes canker disease in many species, and its Pmk1 homolog, CcPmk1, is required for fungal development and pathogenicity. However, the global regulation network of CcPmk1 is still unclear. In this study, we compared transcriptional analysis between a CcPmk1 deletion mutant and the wild type during the simulated infection process. A subset of transcription factor genes and putative effector genes were significantly down-regulated in the CcPmk1 deletion mutant, which might be important for fungal pathogenicity. Additionally, many tandem genes were found to be regulated by CcPmk1. Eleven out of 68 core secondary metabolism biosynthesis genes and several gene clusters were significantly down-regulated in the CcPmk1 deletion mutant. GO annotation of down-regulated genes showed that the ribosome biosynthesis-related processes were over-represented in the CcPmk1 deletion mutant. Comparison of the CcPmk1-regulated genes with the Pmk1-regulated genes from Magnaporthe oryzae revealed only a few overlapping regulated genes in both CcPmk1 and Pmk1, while the enrichment GO terms in the ribosome biosynthesis-related processes were also found. Subsequently, we calculated that in vitro feeding artificial small interference RNAs of CcPmk1 could silence the target gene, resulting in inhibited fungal growth. Furthermore, silencing of BcPmk1 in Botrytis cinerea with conserved CcPmk1 and BcPmk1 fragments could significantly compromise fungal virulence using the virus-induced gene silencing system in Nicotiana benthamiana. These results suggest that CcPmk1 functions as a regulator of pathogenicity and can potentially be designed as a target for broad-spectrum disease control, but unintended effects on nonpathogenic fungi need to be avoided.


Assuntos
Ascomicetos/genética , Botrytis/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Doenças das Plantas/microbiologia , Transdução de Sinais , Árvores/microbiologia , Ascomicetos/patogenicidade , Botrytis/patogenicidade , Regulação para Baixo , Proteínas Fúngicas/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Doenças das Plantas/prevenção & controle , Tabaco/microbiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação para Cima
13.
Sci Rep ; 11(1): 7396, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33795728

RESUMO

Microorganisms that activate plant immune responses have attracted considerable attention as potential biocontrol agents in agriculture because they could reduce agrochemical use. However, conventional methods to screen for such microorganisms using whole plants and pathogens are generally laborious and time consuming. Here, we describe a general strategy using cultured plant cells to identify microorganisms that activate plant defense responses based on plant-microbe interactions. Microbial cells were incubated with tobacco BY-2 cells, followed by treatment with cryptogein, a proteinaceous elicitor of tobacco immune responses secreted by an oomycete. Cryptogein-induced production of reactive oxygen species (ROS) in BY-2 cells served as a marker to evaluate the potential of microorganisms to activate plant defense responses. Twenty-nine bacterial strains isolated from the interior of Brassica rapa var. perviridis plants were screened, and 8 strains that enhanced cryptogein-induced ROS production in BY-2 cells were selected. Following application of these strains to the root tip of Arabidopsis seedlings, two strains, Delftia sp. BR1R-2 and Arthrobacter sp. BR2S-6, were found to induce whole-plant resistance to bacterial pathogens (Pseudomonas syringae pv. tomato DC3000 and Pectobacterium carotovora subsp. carotovora NBRC 14082). Pathogen-induced expression of plant defense-related genes (PR-1, PR-5, and PDF1.2) was enhanced by the pretreatment with strain BR1R-2. This cell-cell interaction-based platform is readily applicable to large-scale screening for microorganisms that enhance plant defense responses under various environmental conditions.


Assuntos
Plantas/imunologia , Plantas/microbiologia , Agricultura , Arabidopsis , Proteínas de Arabidopsis/genética , Brassica rapa/microbiologia , DNA Ribossômico/metabolismo , Endófitos , Proteínas Fúngicas/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas/efeitos dos fármacos , Sistema Imunitário , Filogenia , Células Vegetais/metabolismo , Doenças das Plantas/microbiologia , Imunidade Vegetal/efeitos dos fármacos , Folhas de Planta/metabolismo , Pseudomonas syringae/genética , RNA Ribossômico 16S/metabolismo , Espécies Reativas de Oxigênio , Plântula/metabolismo , Tabaco/microbiologia
14.
Nat Commun ; 12(1): 2166, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846308

RESUMO

Crh proteins catalyze crosslinking of chitin and glucan polymers in fungal cell walls. Here, we show that the BcCrh1 protein from the phytopathogenic fungus Botrytis cinerea acts as a cytoplasmic effector and elicitor of plant defense. BcCrh1 is localized in vacuoles and the endoplasmic reticulum during saprophytic growth. However, upon plant infection, the protein accumulates in infection cushions; it is then secreted to the apoplast and translocated into plant cells, where it induces cell death and defense responses. Two regions of 53 and 35 amino acids are sufficient for protein uptake and cell death induction, respectively. BcCrh1 mutant variants that are unable to dimerize lack transglycosylation activity, but are still able to induce plant cell death. Furthermore, Arabidopsis lines expressing the bccrh1 gene exhibit reduced sensitivity to B. cinerea, suggesting a potential use of the BcCrh1 protein in plant immunization against this necrotrophic pathogen.


Assuntos
Arabidopsis/imunologia , Arabidopsis/microbiologia , Botrytis/enzimologia , Citoplasma/metabolismo , Proteínas Fúngicas/metabolismo , Glicosiltransferases/metabolismo , Células Vegetais/microbiologia , Agrobacterium/metabolismo , Botrytis/crescimento & desenvolvimento , Botrytis/patogenicidade , Morte Celular , Resistência à Doença , Proteínas Fúngicas/química , Doenças das Plantas/microbiologia , Imunidade Vegetal , Multimerização Proteica , Espécies Reativas de Oxigênio/metabolismo , Tabaco/microbiologia
15.
Artigo em Inglês | MEDLINE | ID: mdl-33900905

RESUMO

A novel Gram-negative, facultatively anaerobic, non-spore-forming and rod-shaped bacterial strain (KUDC3025T) was isolated from rhizospheric soil of Artemisia japonica subsp. littoricola collected from the Dokdo Islands, Republic of Korea and bacterial strain MYb239 was isolated from compost from Kiel, Germany. Phylogenetic analysis based on 16S rRNA gene sequences, multilocus genes (atpD, gyrB, infB and rpoB), and whole-genome sequences indicated that both strains belonged to the genus Serratia and were most closely related to Serratia rubidaea KCTC 2927T. The average nucleotide identity values based on blast and MUMmer, tetranucleotide usage pattern and genome-based digital DNA-DNA hybridization values were all below the 95.0 %/95.0 %/0.998/70 % cutoff points. The genome G+C content was 58.0 mol%. The cellular quinone content contained ubiquinone-8 and the major components in the fatty acid profile were C16 : 0, C17 : 0 cyclo and C14 : 0. The polar lipid profile included diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, four unknown amino lipids, two unknown phospholipids and an unknown lipid. Based on phenotypic, chemotaxonomic and genotypic data, strain KUDC3025T (DSM 106578T=CGMCC 1.18473T) and MYb239 represents a novel species, for which the name Serratia rhizosphaerae sp. nov. is proposed. Furthermore, strain KUDC3025T was able to suppress disease symptoms by priming the plant defence system components, including the salicylic acid and ethylene pathways, furthering our understanding of Serratia as potential plant growth promoting bacteria.


Assuntos
Resistência à Doença , Filogenia , Doenças das Plantas/microbiologia , Serratia/classificação , Microbiologia do Solo , Tabaco/microbiologia , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Alemanha , Ilhas , Hibridização de Ácido Nucleico , Fosfolipídeos/química , RNA Ribossômico 16S/genética , República da Coreia , Rizosfera , Análise de Sequência de DNA , Serratia/isolamento & purificação
17.
Biochem Biophys Res Commun ; 554: 7-12, 2021 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-33774281

RESUMO

Agrobacterium T-DNA (transfer DNA) integration into the plant genome relies mostly on host proteins involved in the DNA damage repair pathways. However, conflicting results have been obtained using plants with mutated or down-regulated genes involved in these pathways. Here, we chose a different approach by following the expression of a series of genes, encoding proteins involved in the DNA damage response, during early stages of Agrobacterium infection in tobacco. First, we identified tobacco homologs of Arabidopsis genes induced upon DNA damage and demonstrated that their expression was activated by bleomycin, a DNA-break causing agent. Then, we showed that Agrobacterium infection induces the expression of several of these genes markers of the host DNA damage response, with different patterns of transcriptional response. This induction largely depends on Agrobacterium virulence factors, but not on the T-DNA, suggesting that the DNA damage response activation may rely on Agrobacterium-encoded virulence proteins. Our results suggest that Agrobacterium modulates the plant DNA damage response machinery, which might facilitate the integration of the bacterial T-DNA into the DNA breaks in the host genome.


Assuntos
Agrobacterium tumefaciens/genética , Proteínas de Bactérias/metabolismo , Dano ao DNA , Regulação da Expressão Gênica de Plantas , Tabaco/genética , Fatores de Virulência/metabolismo , Agrobacterium tumefaciens/isolamento & purificação , Agrobacterium tumefaciens/metabolismo , Agrobacterium tumefaciens/patogenicidade , Proteínas de Bactérias/genética , Genes de Plantas , Tabaco/metabolismo , Tabaco/microbiologia , Transformação Genética , Fatores de Virulência/genética
18.
Mol Plant Pathol ; 22(5): 522-538, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33675158

RESUMO

Fusarium oxysporum is an important soilborne fungal pathogen with many different formae speciales that can colonize the plant vascular system and cause serious crop wilt disease worldwide. We found a glycoside hydrolase family 12 protein FoEG1, secreted by F. oxysporum, that acted as a pathogen-associated molecular pattern (PAMP) targeting the apoplast of plants to induce cell death. Purified FoEG1 protein triggered cell death in different plants and induced the plant defence response to enhance the disease resistance of plants. The ability of FoEG1 to induce cell death was mediated by leucine-rich repeat (LRR) receptor-like kinases BAK1 and SOBIR1, and this ability was independent of its hydrolase activity. The mutants of cysteine residues did not affect the ability of FoEG1 to induce cell death, and an 86 amino acid fragment from amino acid positions 144 to 229 of FoEG1 was sufficient to induce cell death in Nicotiana benthamiana. In addition, the expression of FoEG1 was strongly induced in the early stage of F. oxysporum infection of host plants, and FoEG1 deletion or loss of enzyme activity reduced the virulence of F. oxysporum. Therefore, our results suggest that FoEG1 can contribute to the virulence of F. oxysporum depending on its enzyme activity and can also act as a PAMP to induce plant defence responses.


Assuntos
Fusarium/enzimologia , Glicosídeo Hidrolases/metabolismo , Padrões Moleculares Associados a Patógenos/metabolismo , Doenças das Plantas/microbiologia , Imunidade Vegetal , Tabaco/microbiologia , Morte Celular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Fusarium/genética , Fusarium/patogenicidade , Glicosídeo Hidrolases/genética , Doenças das Plantas/imunologia , Tabaco/imunologia , Virulência
19.
Plant Cell ; 33(5): 1447-1471, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-33677602

RESUMO

Pathogens modulate plant cell structure and function by secreting effectors into host tissues. Effectors typically function by associating with host molecules and modulating their activities. This study aimed to identify the host processes targeted by the RXLR class of host-translocated effectors of the potato blight pathogen Phytophthora infestans. To this end, we performed an in planta protein-protein interaction screen by transiently expressing P. infestans RXLR effectors in Nicotiana benthamiana leaves followed by coimmunoprecipitation and liquid chromatography-tandem mass spectrometry. This screen generated an effector-host protein interactome matrix of 59 P. infestans RXLR effectors x 586 N. benthamiana proteins. Classification of the host interactors into putative functional categories revealed over 35 biological processes possibly targeted by P. infestans. We further characterized the PexRD12/31 family of RXLR-WY effectors, which associate and colocalize with components of the vesicle trafficking machinery. One member of this family, PexRD31, increased the number of FYVE positive vesicles in N. benthamiana cells. FYVE positive vesicles also accumulated in leaf cells near P. infestans hyphae, indicating that the pathogen may enhance endosomal trafficking during infection. This interactome dataset will serve as a useful resource for functional studies of P. infestans effectors and of effector-targeted host processes.


Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Phytophthora infestans/fisiologia , Proteínas/metabolismo , Vesículas Transportadoras/metabolismo , Membrana Celular/metabolismo , Endossomos/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Mapas de Interação de Proteínas , Proteínas SNARE/metabolismo , Tabaco/metabolismo , Tabaco/microbiologia
20.
mSphere ; 6(1)2021 02 24.
Artigo em Inglês | MEDLINE | ID: mdl-33627507

RESUMO

Canker disease is caused by the fungus Cytospora chrysosperma and damages a wide range of woody plants, causing major losses to crops and native plants. Plant pathogens secrete virulence-related effectors into host cells during infection to regulate plant immunity and promote colonization. However, the functions of C. chrysosperma effectors remain largely unknown. In this study, we used Agrobacterium tumefaciens-mediated transient expression system in Nicotiana benthamiana and confocal microscopy to investigate the immunoregulation roles and subcellular localization of CcCAP1, a virulence-related effector identified in C. chrysosperma CcCAP1 was significantly induced in the early stages of infection and contains cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins (CAP) superfamily domain with four cysteines. CcCAP1 suppressed the programmed cell death triggered by Bcl-2-associated X protein (BAX) and the elicitin infestin1 (INF1) in transient expression assays with Nicotiana benthamiana The CAP superfamily domain was sufficient for its cell death-inhibiting activity and three of the four cysteines in the CAP superfamily domain were indispensable for its activity. Pathogen challenge assays in N. benthamiana demonstrated that transient expression of CcCAP1 promoted Botrytis cinerea infection and restricted reactive oxygen species accumulation, callose deposition, and defense-related gene expression. In addition, expression of green fluorescent protein-labeled CcCAP1 in N. benthamiana showed that it localized to both the plant nucleus and the cytoplasm, but the nuclear localization was essential for its full immune inhibiting activity. These results suggest that this virulence-related effector of C. chrysosperma modulates plant immunity and functions mainly via its nuclear localization and the CAP domain.IMPORTANCE The data presented in this study provide a key resource for understanding the biology and molecular basis of necrotrophic pathogen responses to Nicotiana benthamiana resistance utilizing effector proteins, and CcCAP1 may be used in future studies to understand effector-triggered susceptibility processes in the Cytospora chrysosperma-poplar interaction system.


Assuntos
Ascomicetos/genética , Ascomicetos/patogenicidade , Núcleo Celular/genética , Proteínas Fúngicas/genética , Imunidade Vegetal , Tabaco/imunologia , Fatores de Virulência/genética , Morte Celular , Doenças das Plantas/microbiologia , Tabaco/genética , Tabaco/microbiologia , Virulência
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