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1.
Plant J ; 118(2): 388-404, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38150324

RESUMO

The intercellular space or apoplast constitutes the main interface in plant-pathogen interactions. Apoplastic subtilisin-like proteases-subtilases-may play an important role in defence and they have been identified as targets of pathogen-secreted effector proteins. Here, we characterise the role of the Solanaceae-specific P69 subtilase family in the interaction between tomato and the vascular bacterial wilt pathogen Ralstonia solanacearum. R. solanacearum infection post-translationally activated several tomato P69s. Among them, P69D was exclusively activated in tomato plants resistant to R. solanacearum. In vitro experiments showed that P69D activation by prodomain removal occurred in an autocatalytic and intramolecular reaction that does not rely on the residue upstream of the processing site. Importantly P69D-deficient tomato plants were more susceptible to bacterial wilt and transient expression of P69B, D and G in Nicotiana benthamiana limited proliferation of R. solanacearum. Our study demonstrates that P69s have conserved features but diverse functions in tomato and that P69D is involved in resistance to R. solanacearum but not to other vascular pathogens like Fusarium oxysporum.


Assuntos
Ralstonia solanacearum , Solanaceae , Solanum lycopersicum , Solanum lycopersicum/genética , Nicotiana/genética , Ralstonia solanacearum/fisiologia , Doenças das Plantas/microbiologia
3.
J Exp Bot ; 72(2): 184-198, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-32976552

RESUMO

Xylem vascular wilt pathogens cause devastating diseases in plants. Proliferation of these pathogens in the xylem causes massive disruption of water and mineral transport, resulting in severe wilting and death of the infected plants. Upon reaching the xylem vascular tissue, these pathogens multiply profusely, spreading vertically within the xylem sap, and horizontally between vessels and to the surrounding tissues. Plant resistance to these pathogens is very complex. One of the most effective defense responses in resistant plants is the formation of physico-chemical barriers in the xylem tissue. Vertical spread within the vessel lumen is restricted by structural barriers, namely, tyloses and gels. Horizontal spread to the apoplast and surrounding healthy vessels and tissues is prevented by vascular coating of the colonized vessels with lignin and suberin. Both vertical and horizontal barriers compartmentalize the pathogen at the infection site and contribute to their elimination. Induction of these defenses are tightly coordinated, both temporally and spatially, to avoid detrimental consequences such as cavitation and embolism. We discuss current knowledge on mechanisms underlying plant-inducible structural barriers against major xylem-colonizing pathogens. This knowledge may be applied to engineer metabolic pathways of vascular coating compounds in specific cells, to produce plants resistant towards xylem colonizers.


Assuntos
Doenças das Plantas , Solanum lycopersicum , Xilema
4.
Sci Rep ; 10(1): 5661, 2020 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-32205847

RESUMO

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

5.
J Exp Bot ; 71(6): 2157-2171, 2020 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-32211785

RESUMO

Ralstonia solanacearum is a bacterial vascular pathogen causing devastating bacterial wilt. In the field, resistance against this pathogen is quantitative and is available for breeders only in tomato and eggplant. To understand the basis of resistance to R. solanacearum in tomato, we investigated the spatio-temporal dynamics of bacterial colonization using non-invasive live monitoring techniques coupled to grafting of susceptible and resistant varieties. We found four 'bottlenecks' that limit the bacterium in resistant tomato: root colonization, vertical movement from roots to shoots, circular vascular bundle invasion, and radial apoplastic spread in the cortex. Radial invasion of cortical extracellular spaces occurred mostly at late disease stages but was observed throughout plant infection. This study shows that resistance is expressed in both root and shoot tissues, and highlights the importance of structural constraints to bacterial spread as a resistance mechanism. It also shows that R. solanacearum is not only a vascular pathogen but spreads out of the xylem, occupying the plant apoplast niche. Our work will help elucidate the complex genetic determinants of resistance, setting the foundations to decipher the molecular mechanisms that limit pathogen colonization, which may provide new precision tools to fight bacterial wilt in the field.


Assuntos
Ralstonia solanacearum , Solanum lycopersicum , Solanum melongena , Doenças das Plantas , Xilema
6.
Sci Rep ; 8(1): 10531, 2018 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-30002392

RESUMO

Phytaspases are Asp-specific subtilisin-like plant proteases that have been likened to animal caspases with respect to their regulatory function in programmed cell death (PCD). We identified twelve putative phytaspase genes in tomato that differed widely in expression level and tissue-specific expression patterns. Most phytaspase genes are tandemly arranged on tomato chromosomes one, four, and eight, and many belong to taxon-specific clades, e.g. the P69 clade in the nightshade family, suggesting that these genes evolved by gene duplication after speciation. Five tomato phytaspases (SlPhyts) were expressed in N. benthamiana and purified to homogeneity. Substrate specificity was analyzed in a proteomics assay and with a panel of fluorogenic peptide substrates. Similar to animal caspases, SlPhyts recognized an extended sequence motif including Asp at the cleavage site. Clear differences in cleavage site preference were observed implying different substrates in vivo and, consequently, different physiological functions. A caspase-like function in PCD was confirmed for five of the seven tested phytaspases. Cell death was triggered by ectopic expression of SlPhyts 2, 3, 4, 5, 6 in tomato leaves by agro-infiltration, as well as in stably transformed transgenic tomato plants. SlPhyts 3, 4, and 5 were found to contribute to cell death under oxidative stress conditions.


Assuntos
Caspases/metabolismo , Proteínas de Plantas/metabolismo , Solanum lycopersicum/metabolismo , Apoptose/fisiologia , Caspases/genética , Morte Celular , Expressão Ectópica do Gene , Duplicação Gênica , Genes de Plantas/genética , Solanum lycopersicum/genética , Estresse Oxidativo/fisiologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Proteômica , Especificidade por Substrato , Nicotiana/genética , Nicotiana/metabolismo
7.
Mol Cell Proteomics ; 17(6): 1112-1125, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29523767

RESUMO

Activity-based protein profiling (ABPP) is a powerful proteomic technique to display protein activities in a proteome. It is based on the use of small molecular probes that react with the active site of proteins in an activity-dependent manner. We used ABPP to dissect the protein activity changes that occur in the intercellular spaces of tolerant (Hawaii 7996) and susceptible (Marmande) tomato plants in response to R. solanacearum, the causing agent of bacterial wilt, one of the most destructive bacterial diseases in plants. The intercellular space -or apoplast- is the first battlefield where the plant faces R. solanacearum Here, we explore the possibility that the limited R. solanacearum colonization reported in the apoplast of tolerant tomato is partly determined by its active proteome. Our work reveals specific activation of papain-like cysteine proteases (PLCPs) and serine hydrolases (SHs) in the leaf apoplast of the tolerant tomato Hawaii 7996 on R. solanacearum infection. The P69 family members P69C and P69F, and an unannotated lipase (Solyc02g077110.2.1), were found to be post-translationally activated. In addition, protein network analysis showed that deeper changes in network topology take place in the susceptible tomato variety, suggesting that the tolerant cultivar might be more prepared to face R. solanacearum in its basal state. Altogether this work identifies significant changes in the activity of 4 PLCPs and 27 SHs in the tomato leaf apoplast in response to R. solanacearum, most of which are yet to be characterized. Our findings denote the importance of novel proteomic approaches such as ABPP to provide new insights on old and elusive questions regarding the molecular basis of resistance to R. solanacearum.


Assuntos
Peptídeo Hidrolases/metabolismo , Doenças das Plantas , Proteínas de Plantas/metabolismo , Ralstonia solanacearum , Solanum lycopersicum/metabolismo , Resistência à Doença/fisiologia , Solanum lycopersicum/microbiologia
8.
Mol Plant Microbe Interact ; 31(1): 175-184, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-28840786

RESUMO

The causal agent of bacterial wilt, Ralstonia solanacearum, is a soilborne pathogen that invades plants through their roots, traversing many tissue layers until it reaches the xylem, where it multiplies and causes plant collapse. The effects of R. solanacearum infection are devastating, and no effective approach to fight the disease is so far available. The early steps of infection, essential for colonization, as well as the early plant defense responses remain mostly unknown. Here, we have set up a simple, in vitro Arabidopsis thaliana-R. solanacearum pathosystem that has allowed us to identify three clear root phenotypes specifically associated to the early stages of infection: root-growth inhibition, root-hair formation, and root-tip cell death. Using this method, we have been able to differentiate, on Arabidopsis plants, the phenotypes caused by mutants in the key bacterial virulence regulators hrpB and hrpG, which remained indistinguishable using the classical soil-drench inoculation pathogenicity assays. In addition, we have revealed the previously unknown involvement of auxins in the root rearrangements caused by R. solanacearum infection. Our system provides an easy-to-use, high-throughput tool to study R. solanacearum aggressiveness. Furthermore, the observed phenotypes may allow the identification of bacterial virulence determinants and could even be used to screen for novel forms of early plant resistance to bacterial wilt.


Assuntos
Arabidopsis/microbiologia , Proteínas de Bactérias/metabolismo , Sistemas de Secreção Bacterianos , Raízes de Plantas/microbiologia , Ralstonia solanacearum/metabolismo , Resistência à Doença , Mutação/genética , Fenótipo , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Raízes de Plantas/crescimento & desenvolvimento , Ralstonia solanacearum/patogenicidade , Virulência
9.
Methods Mol Biol ; 1450: 195-203, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27424755

RESUMO

Plants are constantly exposed to a complex and changing environment that challenges their cellular homeostasis. Stress responses triggered as a consequence of unfavorable conditions result in increased protein aggregate formation at the cellular level. When the formation of misfolded proteins surpasses the capacity of the cell to remove them, insoluble protein aggregates accumulate. In the animal field, an enormous effort is being placed to uncover the mechanisms regulating aggregate formation because of its implications in many important human diseases. Because of its importance for cellular functionality and fitness, it is equally important to expand plant research in this field. Here, we describe a cell fractionation-based method to obtain very pure insoluble protein aggregate fractions that can be subsequently semiquantified using image analysis. This method can be used as a first step to evaluate whether a particular condition results in an alteration of protein aggregate formation levels.


Assuntos
Biologia Molecular/métodos , Proteínas de Plantas/isolamento & purificação , Agregados Proteicos/genética , Humanos , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas/química , Plantas/genética , Biossíntese de Proteínas/genética
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