Apoplastic Fluid Research Articles

Identification and characterization of a serine protease from wheat leaves

A putative serine protease with a potential role in the plant biotic and abiotic stress response was purified from wheat leaf apoplastic fluid and partially characterized. Following two-dimensional electrophoresis a protein of Mr = 75 k and a pI of 4.2 to 4.5 was observed. This protein displayed in-gel protease activity and was specifically inhibited by phenylmethanesulfonyl fluoride and partially inhibited by Ca2+ and Zn2+, but not by E-64 or leupeptin. An internal tryptic fragment of 13 amino acids was identified by MALDI QqTOF MS/MS, and this peptide showed a high level of homology (85–100 % identity) to a highly conserved region of known plant subtilisin-like proteases. We demonstrated that the protease activity increased until a late stage of wheat leaf development and increased in response to heat shock. In both cases Rubisco large subunit was degraded with time. Protease activity was also increased during biotic stress. Leaves challenged with leaf rust (Puccinia triticina), showed an approximately three fold increase in protease activity during an incompatible interaction, compared to activity in mock-inoculated leaves and to leaves in a compatible leaf rust interaction. These results suggest that the expression of this serine protease could be involved in the defense response against both abiotic and biotic stresses.

Collection of Apoplastic Fluids from Arabidopsis thaliana Leaves.

The leaf apoplast comprises the extracellular continuum outside cell membranes. A broad range of processes take place in the apoplast, including intercellular signaling, metabolite transport, and plant-microbe interactions. To study these processes, it is essential to analyze the metabolite content in apoplastic fluids. Due to the fragile nature of leaf tissues, it is a challenge to obtain apoplastic fluids from leaves. Here, methods to collect apoplastic washing fluid and guttation fluid from Arabidopsis thaliana leaves are described.

Isolation of Exosome-Like Vesicles from Plants by Ultracentrifugation on Sucrose/Deuterium Oxide (D2O) Density Cushions.

Exosomes are nanovesicles of endocytic origin that are about 30-100 nm in diameter, surrounded by a lipid bilayer membrane, and contain proteins, nucleic acids, and other molecules. Mammalian cells- and biological fluids-derived exosomes have become the subject for a wide range of investigations in biological and biomedical sciences. More recently, a new interest is on the verge of rising: the presence of nanovesicles in plants. Lipoprotein vesicles from apoplastic fluid and exosome-like vesicles (ELVs) from fruit juice have been isolated and shown that they could be loaded with drugs and uptaken by recipient cells. In order to explore and analyze the contents and functions of ELVs, they must be isolated and purified with intense care. Isolation of ELVs can be a tedious process and often characterized by the co-purification of undesired contaminants. Here we describe a method which isolates ELVs based on their buoyant density. The method utilizes differential centrifugation in step 1 and 1 and 2 M sucrose/deuterium oxide double-cushion ultracentrifugation in step 2, to purify two diverse ELV subpopulations. In this method fruit juice is used as an example of starting material, although this protocol can be used for the isolation of vesicles from apoplastic fluid too. The quality and the quantity of ELV preparations have been found appropriate for downstream biological and structural studies, like proteomics, transcriptomics, and lipidomics.

Vacuum Infiltration-Centrifugation Method for Apoplastic Protein Extraction in Grapevine.

The apoplastic fluid moving in the extracellular space external to the plasma membrane provides a means of delivering molecules and facilitates intercellular communications. However, the apoplastic fluid extraction from in planta systems remains challenging and this is particularly true for grapevine (Vitis vinifera L.), a worldwide-cultivated fruit plant. Here, we describe an optimized vacuum-infiltration-centrifugation method to extract soluble proteins from apoplastic fluid of grapevine leaves. This optimized method allows recovering of the grapevine apoplastic soluble proteins suitable for mono- and bi-dimensional gel electrophoresis for further proteomic analysis in order to elucidate their physiological functions.

TcCYPR04, a Cacao Papain-Like Cysteine-Protease Detected in Senescent and Necrotic Tissues Interacts with a Cystatin TcCYS4.

The interaction amongst papain-like cysteine-proteases (PLCP) and their substrates and inhibitors, such as cystatins, can be perceived as part of the molecular battlefield in plant-pathogen interaction. In cacao, four cystatins were identified and characterized by our group. We identified 448 proteases in cacao genome, whereof 134 were cysteine-proteases. We expressed in Escherichia coli a PLCP from cacao, named TcCYSPR04. Immunoblottings with anti-TcCYSPR04 exhibited protein increases during leaf development. Additional isoforms of TcCYSPR04 appeared in senescent leaves and cacao tissues infected by Moniliophthora perniciosa during the transition from the biotrophic to the saprophytic phase. TcCYSPR04 was induced in the apoplastic fluid of Catongo and TSH1188 cacao genotypes, susceptible and resistant to M. perniciosa, respectively, but greater intensity and additional isoforms were observed in TSH1188. The fungal protein MpNEP induced PLCP isoform expression in tobacco leaves, according to the cross reaction with anti-TcCYSPR04. Several protein isoforms were detected at 72 hours after treatment with MpNEP. We captured an active PLCP from cacao tissues, using a recombinant cacao cystatin immobilized in CNBr-Sepharose. Mass spectrometry showed that this protein corresponds to TcCYSPR04. A homology modeling was obtained for both proteins. In order to become active, TcCYSPR04 needs to lose its inhibitory domain. Molecular docking showed the physical-chemical complementarities of the interaction between the cacao enzyme and its inhibitor. We propose that TcCYSPR04 and its interactions with cacao cystatins are involved in the senescence and necrosis events related to witches’ broom symptoms. This molecular interaction may be the target for future interventions to control witches' broom disease.

Integrated management of Fusarium crown rot of wheat using fungicide seed treatment, cultivar resistance, and induction of systemic acquired resistance (SAR)

Fusarium crown rot (FCR) of wheat (Triticum aestivum L.) is a perennial problem for wheat producers worldwide. Integration of the different management tools was assessed to determine a new strategy to reduce FCR. Difenoconazole–mefenoxam fungicide seed treatment reduced FCR severity 29–50% in glasshouse trials, while seed treatment with Bacillus pumilis isolate 314-16-5 and Trichoderma harzianum T-22 provided control statistically different than the untreated or seed treated with Bacillus isolate L324-92 or Pseudomonas fluorescens isolate 2-79. Assessment of resistance in inoculated glasshouse trials and innate activity levels of three pathogenesis-related (PR) proteins in apoplastic fluids of five non-inoculated spring wheat cultivars showed the lowest disease severity on cv. Volt and the highest levels of endochitinase and β-1,3-glucanase activity compared to the other cultivars (P<0.05). Induction of SAR with foliar applications of Bacillus mycoides isolate BmJ (1.5×108cfu/ml) or acibenzolar-S-methyl (ASM, 1.0mM) on the cultivars Hank, Knudson and Volt reduced the severity of FCR by 10% compared to a water control (P<0.05). BmJ application increased concentrations of peroxidase and endochitinase, while ASM increased β-1,3-glucanases levels in cultivars Volt and Hank compared to water controls (P<0.05). Integration of the management tools, difenoconazole–mefenoxam seed treatment, cultivar resistance, and SAR induction, showed integration of all three management tools did not reduce disease severity more than use of cultivar resistance plus fungicide seed treatment or SAR induction in glasshouse trials. In a dryland field trial, integration of all three management tools reduced disease severity and FCR populations more than individual tools (P<0.05).

Toward the Identification of Two Glycoproteins Involved in the Stomatal Deregulation of Downy Mildew-Infected Grapevine Leaves.

Stomata remain abnormally opened and unresponsive to abscisic acid in grapevine leaves infected by downy mildew. This deregulation occurs from 3 days postinoculation and increases concomitantly with leaf colonization by the pathogen. Using epidermal peels, we demonstrated that the active compound involved in this deregulation is located in the apoplast. Biochemical assays showed that the active compound present in the apoplastic fluids isolated from Plasmopara viticola-infected grapevine leaves (IAF) is a CysCys bridge-independent, thermostable and glycosylated protein. Fractionation guided assays based on chromatography coupled to stomatal response and proteomic analysis allowed the identification of both plant and pathogen proteins in the active fraction obtained from IAF. Further in silico analysis and discriminant filtrations based on the comparison between predictions and experimental indications lead to the identification of two Vitis vinifera proteins as candidates for the observed stomatal deregulation.

Isolation of novel leaf-inhabiting endophytic bacteria in Arabidopsis thaliana and their antagonistic effects on phytophathogens

Bacterial endophytes benefit the host with protection against abiotic and biotic stresses and through increased plant growth. In this study, we screened novel endophytes for the ability to endophytically inhabit leaf tissues. Four endophytic bacteria were screened from the apoplastic fluids extracted from the uninfected upper leaves of Arabidopsis thaliana 3 days postinoculation (dpi) with Pseudomonas syringae pv. tomato DC3000 (Pst). Under sterile plant growth conditions, we showed that the four isolates proliferated intercellularly in the leaf tissues of A. thaliana. Based on 16S rRNA sequencing analysis, the identities of the inoculated endophytes and the re-isolates from the leaf tissues were confirmed. Among the isolates, the two species of Rhodococcus were the first members of the genus to be identified as leaf-inhabiting endophytes. Additionally, three of the isolates showed antagonistic activities, with different levels of activity, against Fusarium oxysporum pv. conglutinans (F.o.) and/or Pst. Furthermore, the application of one isolate, Bacillus cereus KB1, on tomato plants significantly increased the disease resistance to Botrytis cinerea and Pst. In combination, these results indicate that these endophytic isolates can be used to develop potential biocontrol agents against a variety of pathogenic fungi and bacteria.

Persistence of the Host-Selective Toxin Ptr ToxB in the Apoplast.

The necrotrophic fungus Pyrenophora tritici-repentis is responsible for the disease tan spot of wheat. Ptr ToxB (ToxB), a proteinaceous host-selective toxin, is one of the effectors secreted by P. tritici-repentis. ToxB induces chlorosis in toxin-sensitive wheat cultivars and displays characteristics common to apoplastic effectors. We addressed the hypothesis that ToxB exerts its activity extracellularly. Our data indicate that hydraulic pressure applied in the apoplast following ToxB infiltration can displace ToxB-induced symptoms. In addition, treatment with a proteolytic cocktail following toxin infiltration results in reduction of symptom development and indicates that ToxB requires at least 8 h in planta to induce maximum symptom development. In vitro assays demonstrate that apoplastic fluids extracted from toxin-sensitive and -insensitive wheat cultivars cannot degrade ToxB. Additionally, ToxB can be reisolated from apoplastic fluid after toxin infiltration. Furthermore, localization studies of fluorescently labeled ToxB indicate that the toxin remains in the apoplast in toxin-sensitive and -insensitive wheat cultivars. Our findings support the hypothesis that ToxB acts as an extracellular effector.

The Identification of Genes Important in Pseudomonas syringae pv. phaseolicola Plant Colonisation Using In Vitro Screening of Transposon Libraries.

The bacterial plant pathogen Pseudomonas syringae pv. phaseolicola (Pph) colonises the surface of common bean plants before moving into the interior of plant tissue, via wounds and stomata. In the intercellular spaces the pathogen proliferates in the apoplastic fluid and forms microcolonies (biofilms) around plant cells. If the pathogen can suppress the plant’s natural resistance response, it will cause halo blight disease. The process of resistance suppression is fairly well understood, but the mechanisms used by the pathogen in colonisation are less clear. We hypothesised that we could apply in vitro genetic screens to look for changes in motility, colony formation, and adhesion, which are proxies for infection, microcolony formation and cell adhesion. We made transposon (Tn) mutant libraries of Pph strains 1448A and 1302A and found 106/1920 mutants exhibited alterations in colony morphology, motility and biofilm formation. Identification of the insertion point of the Tn identified within the genome highlighted, as expected, a number of altered motility mutants bearing mutations in genes encoding various parts of the flagellum. Genes involved in nutrient biosynthesis, membrane associated proteins, and a number of conserved hypothetical protein (CHP) genes were also identified. A mutation of one CHP gene caused a positive increase in in planta bacterial growth. This rapid and inexpensive screening method allows the discovery of genes important for in vitro traits that can be correlated to roles in the plant interaction.

Accumulation of cynaropicrin in globe artichoke and localization of enzymes involved in its biosynthesis

Globe artichoke (Cynara cardunculus var. scolymus) belongs to the Asteraceae family, in which one of the most biologically significant class of secondary metabolites are sesquiterpene lactones (STLs). In globe artichoke the principal STL is the cynaropicrin, which contributes to approximately 80% of its characteristic bitter taste. Cynaropicrin content was assessed in globe artichoke tissues and was observed to accumulate in leaves of different developmental stages. In the receptacle, a progressive decrease was observed during inflorescence development, while the STL could not be detected in the inflorescence bracts. Almost undetectable amounts were found in the roots and inflorescence stems at the commercial stage. Cynaropicrin content was found to correlate with expression of genes encoding CcGAS, CcGAO and CcCOS, which are involved in the STL biosynthesis.A more detailed study of leaf material revealed that cynaropicrin predominantly accumulates in the trichomes, and not in the apoplastic cavity fluids. Analysis of the promoter regions of CcGAO and CcCOS revealed the presence of L1-box motifs, which confers trichome-specific expression in Arabidopsis, suggesting that cynaropicrin is not only stored but also synthesized in trichomes. A transient expression of GFP fusion proteins was performed in Nicotiana benthamiana plants: the CcGAS fluorescence signal was located in the cytoplasm while the CcGAO and CcCOS localized to the endoplasmatic reticulum.

Proteomic analysis of apoplastic fluid of Coffea arabica leaves highlights novel biomarkers for resistance against Hemileia vastatrix.

A proteomic analysis of the apoplastic fluid (APF) of coffee leaves was conducted to investigate the cellular processes associated with incompatible (resistant) and compatible (susceptible) Coffea arabica-Hemileia vastatrix interactions, during the 24–96 hai period. The APF proteins were extracted by leaf vacuum infiltration and protein profiles were obtained by 2-DE. The comparative analysis of the gels revealed 210 polypeptide spots whose volume changed in abundance between samples (control, resistant and susceptible) during the 24–96 hai period. The proteins identified were involved mainly in protein degradation, cell wall metabolism and stress/defense responses, most of them being hydrolases (around 70%), particularly sugar hydrolases and peptidases/proteases. The changes in the APF proteome along the infection process revealed two distinct phases of defense responses, an initial/basal one (24–48 hai) and a late/specific one (72–96 hai). Compared to susceptibility, resistance was associated with a higher number of proteins, which was more evident in the late/specific phase. Proteins involved in the resistance response were mainly, glycohydrolases of the cell wall, serine proteases and pathogen related-like proteins (PR-proteins), suggesting that some of these proteins could be putative candidates for resistant markers of coffee to H. vastatrix. Antibodies were produced against chitinase, pectin methylesterase, serine carboxypeptidase, reticuline oxidase and subtilase and by an immunodetection assay it was observed an increase of these proteins in the resistant sample. With this methodology we have identified proteins that are candidate markers of resistance and that will be useful in coffee breeding programs to assist in the selection of cultivars with resistance to H. vastatrix.

Proteome catalog of Zymoseptoria tritici captured during pathogenesis in wheat

Zymoseptoria tritici is an economically important pathogen of wheat. However, the molecular basis of pathogenicity on wheat is still poorly understood. Here, we present a global survey of the proteins secreted by this fungus in the apoplast of resistant (cv. Shafir) and susceptible (cv. Obelisk) wheat cultivars after inoculation with reference Z. tritici strain IPO323. The fungal proteins present in apoplastic fluids were analyzed by gel electrophoresis and by data-independent acquisition liquid chromatography/mass spectrometry (LC/MSE) combined with data-dependent acquisition LC–MS/MS. Subsequent mapping mass spectrometry-derived peptide sequence data against the genome sequence of strain IPO323 identified 665 peptides in the MSE and 93 in the LC–MS/MS mode that matched to 85 proteins. The identified fungal proteins, including cell-wall degrading enzymes and proteases, might function in pathogenicity, but the functions of many remain unknown. Most fungal proteins accumulated in cv. Obelisk at the onset of necrotrophy. This inventory provides an excellent basis for future detailed studies on the role of these genes and their encoded proteins during pathogenesis in wheat.

Unraveling incompatibility between wheat and the fungal pathogen Zymoseptoria tritici through apoplastic proteomics.

BackgroundHemibiotrophic fungal pathogen Zymoseptoria tritici causes severe foliar disease in wheat. However, current knowledge of molecular mechanisms involved in plant resistance to Z. tritici and Z. tritici virulence factors is far from being complete. The present work investigated the proteome of leaf apoplastic fluid with emphasis on both host wheat and Z. tritici during the compatible and incompatible interactions.ResultsThe proteomics analysis revealed rapid host responses to the biotrophic growth, including enhanced carbohydrate metabolism, apoplastic defenses and stress, and cell wall reinforcement, might contribute to resistance. Compatibility between the host and the pathogen was associated with inactivated plant apoplastic responses as well as fungal defenses to oxidative stress and perturbation of plant cell wall during the initial biotrophic stage, followed by the strong induction of plant defenses during the necrotrophic stage. To study the role of anti-oxidative stress in Z. tritici pathogenicity in depth, a YAP1 transcription factor regulating antioxidant expression was deleted and showed the contribution to anti-oxidative stress in Z. tritici, but was not required for pathogenicity. This result suggests the functional redundancy of antioxidants in the fungus.ConclusionsThe data demonstrate that incompatibility is probably resulted from the proteome-level activation of host apoplastic defenses as well as fungal incapability to adapt to stress and interfere with host cell at the biotrophic stage of the interaction.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1549-6) contains supplementary material, which is available to authorized users.

Protein profile of Beta vulgaris leaf apoplastic fluid and changes induced by Fe deficiency and Fe resupply.

The fluid collected by direct leaf centrifugation has been used to study the proteome of the sugar beet apoplastic fluid as well as the changes induced by Fe deficiency and Fe resupply to Fe-deficient plants in the protein profile. Plants were grown in Fe-sufficient and Fe-deficient conditions, and Fe resupply was carried out with 45 μM Fe(III)-EDTA for 24 h. Protein extracts of leaf apoplastic fluid were analyzed by two-dimensional isoelectric focusing-SDS-PAGE electrophoresis. Gel image analysis revealed 203 consistent spots, and proteins in 81% of them (164) were identified by nLC-MS/MS using a custom made reference repository of beet protein sequences. When redundant UniProt entries were deleted, a non-redundant leaf apoplastic proteome consisting of 109 proteins was obtained. TargetP and SecretomeP algorithms predicted that 63% of them were secretory proteins. Functional classification of the non-redundant proteins indicated that stress and defense, protein metabolism, cell wall and C metabolism accounted for approximately 75% of the identified proteome. The effects of Fe-deficiency on the leaf apoplast proteome were limited, with only five spots (2.5%) changing in relative abundance, thus suggesting that protein homeostasis in the leaf apoplast fluid is well-maintained upon Fe shortage. The identification of three chitinase isoforms among proteins increasing in relative abundance with Fe-deficiency suggests that one of the few effects of Fe deficiency in the leaf apoplast proteome includes cell wall modifications. Iron resupply to Fe deficient plants changed the relative abundance of 16 spots when compared to either Fe-sufficient or Fe-deficient samples. Proteins identified in these spots can be broadly classified as those responding to Fe-resupply, which included defense and cell wall related proteins, and non-responsive, which are mainly protein metabolism related proteins and whose changes in relative abundance followed the same trend as with Fe-deficiency.

Extraction of Apoplastic Wash Fluids and Leaf Petiole Exudates from Leaves of Arabidopsis thaliana

The long-distance translocation of metabolites and mineral elements is crucial for plant growth and reproduction. In most cases, source-to-sink translocation of metabolites and minerals requires their passage through the apoplast, irrespective whether they are transported via the xylem or the phloem. This apoplast-mediated pathway is of particular importance during plant senescence, when photoassimilates as well as organic, inorganic or chelated forms of nutrients are translocated from leaves to fruits or seeds. Recent genetic and physiological studies revealed the involvement of numerous membrane transporters mediating phloem loading of amino acids, sugars, urea or mineral elements. To evaluate the contribution of individual transporters to xylem unloading or phloem loading, the collection of apoplastic fluids and of phloem sap is essential. Here, we describe a method for the extraction of apoplastic fluids and the collection of leaf petiole exudates from Arabidopsis leaves, the latter representing an approximation to the real composition of the phloem sap.

Effect of silencing of the apoplastic invertase gene on photosynthesis in tomato

Photosynthesis was examined in wild-type tomato plants (Lycopersicon esculentum L., cv. Moneymaker) and in the transformants where gene expression of the leaf apoplastic invertase was suppressed by RNA interference (Lin8-RNAi). The influence of genetic transformation on photosynthesis depended on the demand for assimilates by sink organs. Using growth pots with low amount of soil, we found that at the initial growth stage when growth processes were particularly active, photosynthesis in Lin8-RNAi plants was higher than in the wild-type plants. As the reserves of mineral nutrients were gradually exhausted, photosynthesis decreased in both plant groups, but the decrease was more extensive in Lin8-RNAi plant form. Analysis of the distribution of 14C among the photosynthates produced after 3-min period of 14CO2 assimilation revealed the decreased incorporation of 14C into hexoses in Lin8-RNAi plants and the increased incorporation of 14C into aspartate and products of the glycolate pathway. Supplementing the soil with nitrate nitrogen as a fertilizer enhanced the non-carbohydrate trend of photosynthesis, but this trend was less pronounced in the transformed plants. Simultaneous measurements of CO2-exchange and H2O release revealed an insignificant increase in Lin8-RNAi plants of photosynthetic activity, transpiration, and intraleaf CO2 concentration. However, in 30–50 min after lowering the photon flux density from 1556 to 771 μmol/(m2 s) photosynthesis was reduced in both genotypes, whereas transpiration was diminished in wild-type plants and increased in the Lin8-RNAi form. It is concluded that the apoplastic invertase regulates photosynthesis through changes in osmolarity of the apoplastic fluid that controls the opening of stomata.

Apoplastic and intracellular plant sugars regulate developmental transitions in witches' broom disease of cacao.

Witches' broom disease (WBD) of cacao differs from other typical hemibiotrophic plant diseases by its unusually long biotrophic phase. Plant carbon sources have been proposed to regulate WBD developmental transitions; however, nothing is known about their availability at the plant-fungus interface, the apoplastic fluid of cacao. Data are provided supporting a role for the dynamics of soluble carbon in the apoplastic fluid in prompting the end of the biotrophic phase of infection. Carbon depletion and the consequent fungal sensing of starvation were identified as key signalling factors at the apoplast. MpNEP2, a fungal effector of host necrosis, was found to be up-regulated in an autophagic-like response to carbon starvation in vitro. In addition, the in vivo artificial manipulation of carbon availability in the apoplastic fluid considerably modulated both its expression and plant necrosis rate. Strikingly, infected cacao tissues accumulated intracellular hexoses, and showed stunted photosynthesis and the up-regulation of senescence markers immediately prior to the transition to the necrotrophic phase. These opposite findings of carbon depletion and accumulation in different host cell compartments are discussed within the frame of WBD development. A model is suggested to explain phase transition as a synergic outcome of fungal-related factors released upon sensing of extracellular carbon starvation, and an early senescence of infected tissues probably triggered by intracellular sugar accumulation.

The infiltration-centrifugation technique for extraction of apoplastic fluid from plant leaves using Phaseolus vulgaris as an example.

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.

The Infiltration-centrifugation Technique for Extraction of Apoplastic Fluid from Plant Leaves Using &lt;em&gt;Phaseolus vulgaris&lt;/em&gt; as an Example

The apoplast is a distinct extracellular compartment in plant tissues that lies outside the plasma membrane and includes the cell wall. The apoplastic compartment of plant leaves is the site of several important biological processes, including cell wall formation, cellular nutrient and water uptake and export, plant-endophyte interactions and defence responses to pathogens. The infiltration-centrifugation method is well established as a robust technique for the analysis of the soluble apoplast composition of various plant species. The fluid obtained by this method is commonly known as apoplast washing fluid (AWF). The following protocol describes an optimized vacuum infiltration and centrifugation method for AWF extraction from Phaseolus vulgaris (French bean) cv. Tendergreen leaves. The limitations of this method and the optimization of the protocol for other plant species are discussed. Recovered AWF can be used in a wide range of downstream experiments that seek to characterize the composition of the apoplast and how it varies in response to plant species and genotype, plant development and environmental conditions, or to determine how microorganisms grow in apoplast fluid and respond to changes in its composition.