Haustorial Complexes Research Articles

The powdery mildew resistance protein RPW8.2 is carried on VAMP721/722 vesicles to the extrahaustorial membrane of haustorial complexes.

Plants employ multiple cell-autonomous defense mechanisms to impede pathogenesis of microbial intruders. Previously we identified an exocytosis defense mechanism in Arabidopsis against pathogenic powdery mildew fungi. This pre-invasive defense mechanism depends on the formation of ternary protein complexes consisting of the plasma membrane-localized PEN1 syntaxin, the adaptor protein SNAP33 and closely sequence-related vesicle-resident VAMP721 or VAMP722 proteins. The Arabidopsis thaliana resistance to powdery mildew 8.2 protein (RPW8.2) confers disease resistance against powdery mildews upon fungal entry into host cells and is specifically targeted to the extrahaustorial membrane (EHM), which envelops the haustorial complex of the fungus. However, the secretory machinery involved in trafficking RPW8.2 to the EHM is unknown. Here we report that RPW8.2 is transiently located on VAMP721/722 vesicles, and later incorporated into the EHM of mature haustoria. Resistance activity of RPW8.2 against the powdery mildew Golovinomyces orontii is greatly diminished in the absence of VAMP721 but only slightly so in the absence of VAMP722. Consistent with this result, trafficking of RPW8.2 to the EHM is delayed in the absence of VAMP721. These findings implicate VAMP721/722 vesicles as key components of the secretory machinery for carrying RPW8.2 to the plant-fungal interface. Quantitative fluorescence recovery after photobleaching suggests that vesicle-mediated trafficking of RPW8.2-yellow fluorescent protein (YFP) to the EHM occurs transiently during early haustorial development and that lateral diffusion of RPW8.2-YFP within the EHM exceeds vesicle-mediated replenishment of RPW8.2-YFP in mature haustoria. Our findings imply the engagement of VAMP721/722 in a bifurcated trafficking pathway for pre-invasive defense at the cell periphery and post-invasive defense at the EHM.

First catalogue of the rust fungi of French Guiana, northern South America

An annotated list of the rust fungi (Uredinales or Pucciniales) of French Guiana is presented. It enumerates 68 species of which 57 are new reports for the department and 3, Aecidium plukenetiae, Puccinia kourouensis and P. parianicola, are new to science. Dicheirinia guianensis and Hapalophragmium angylocalycis are excluded from the French Guianan mycobiota. New host plants are reported for Batistopsora crucis-filii, B. pistila, Cerotelium ficicola, C. sabiceae, Crossopsora piperis, Desmella aneimiae, Endophyllum guttatum, Kweilingia divina, Puccinia lateritia, Uredo anthurii and Uromyces anguriae. Previously undescribed characters are presented for Achrotelium lucumae, Chaconia ingae, Cerotelium sabiceae, Prospodium amapaensis, Sphenospora smilacina and Uromyces wulffiae-stenoglossae. Chaconia ingae showed haustorial complexes comprising both intracellular hyphae and D-haustoria. In Cerotelium sabiceae, the haustorial mother cells retained the nuclei while D-haustoria were enucleate. The occurrence of these haustorial types in tropical rust fungi is discussed. Internal basidium formation is described for the first time in Sphenospora: teliospores of S. smilacina produced external or internal basidia. The species richness and composition of the French Guianan rust mycobiota are discussed in a neotropical context.

Transcriptome analysis of enriched Golovinomyces orontii haustoria by deep 454 pyrosequencing

Powdery mildews are phytopathogenic ascomycetes that have an obligate biotrophic lifestyle and establish intimate relationships with their plant hosts. A crucial aspect of this plant–fungus interaction is the formation of specialized fungal infection structures termed haustoria. Although located within the cell boundaries of plant epidermal cells, haustoria remain separated from the plant cytoplasm by a host plasma membrane derivative, the extrahaustorial membrane. Haustoria are thought to represent pivotal sites of nutrient uptake and effector protein delivery. We enriched haustorial complexes from Arabidopsis thaliana plants infected with the powdery mildew fungus Golovinomyces orontii and performed in-depth transcriptome analysis by 454-based pyrosequencing of haustorial cDNAs. We assembled 7077 expressed sequence tag (EST) contigs with greater than 5-fold average coverage and analyzed these with regard to the respective predicted protein functions. We found that transcripts coding for gene products with roles in protein turnover, detoxification of reactive oxygen species and fungal pathogenesis are abundant in the haustorial EST contigs, while surprisingly transcripts encoding presumptive nutrient transporters were not highly represented in the haustorial cDNA library. A substantial proportion (∼38%) of transcripts coding for predicted secreted proteins comprises effector candidates. Our data provide valuable insights into the transcriptome of the key infection structure of a model obligate biotrophic phytopathogen.

Synthesis of C-glycosyl flavonoid phytoalexins as a site-specific response to fungal penetration in cucumber

Leaf tissue harvested from cucumber plants (Cucumis sativus L.) expressing induced resistance against the powdery mildew fungus Podosphaera xanthii (syn. Sphaerotheca fuliginea, Castagne; Braun and Shishkoff) was extracted and analyzed for phytoalexin compounds. Fluorescence microscopy was then used to observe the production of these compounds in planta, and laser scanning confocal microscopy observations were made to locate the subcellular sites of phytoalexin accumulation. Phytochemical analyses and fluorescence microscopy observations revealed the production of autofluorescent C-glycosyl flavonoid phytoalexins within the epidermal tissues of disease-resistant plants undergoing fungal ingress. Phytoalexin production was triggered by the combination of an eliciting/inoculation treatment, and tissue autofluorescence of color characteristic of the phytoalexins reached a maximum 48 h after elicitation prior to subsiding following the collapse of the pathogen. After a second eliciting treatment, disease-resistant plants produced phytoalexins more rapidly in response to fungal challenge. At the cellular level, autofluorescent C-glycosyl flavonoid phytoalexins were observed associated with the plasma membrane of infected epidermal cells immediately following elicitation. In the hours that preceded the collapse of conidial chains, phytoalexins accumulated inside the haustorial complexes of the pathogen within the epidermal cells of disease-resistant plants. Taken together, the results of this study show the timely synthesis of C-glycosyl flavonoid phytoalexins at precise subcellular locations as a key defense reaction used by cucumber to create incompatible interactions with powdery mildew.

Synthesis of C-glycosyl flavonoid phytoalexins as a site-specific response to fungal penetration in cucumber

Leaf tissue harvested from cucumber plants ( Cucumis sativus L.) expressing induced resistance against the powdery mildew fungus Podosphaera xanthii (syn. Sphaerotheca fuliginea, Castagne; Braun and Shishkoff) was extracted and analyzed for phytoalexin compounds. Fluorescence microscopy was then used to observe the production of these compounds in planta, and laser scanning confocal microscopy observations were made to locate the subcellular sites of phytoalexin accumulation. Phytochemical analyses and fluorescence microscopy observations revealed the production of autofluorescent C-glycosyl flavonoid phytoalexins within the epidermal tissues of disease-resistant plants undergoing fungal ingress. Phytoalexin production was triggered by the combination of an eliciting/inoculation treatment, and tissue autofluorescence of color characteristic of the phytoalexins reached a maximum 48 h after elicitation prior to subsiding following the collapse of the pathogen. After a second eliciting treatment, disease-resistant plants produced phytoalexins more rapidly in response to fungal challenge. At the cellular level, autofluorescent C-glycosyl flavonoid phytoalexins were observed associated with the plasma membrane of infected epidermal cells immediately following elicitation. In the hours that preceded the collapse of conidial chains, phytoalexins accumulated inside the haustorial complexes of the pathogen within the epidermal cells of disease-resistant plants. Taken together, the results of this study show the timely synthesis of C-glycosyl flavonoid phytoalexins at precise subcellular locations as a key defense reaction used by cucumber to create incompatible interactions with powdery mildew.

An Arabidopsis Callose Synthase, GSL5, Is Required for Wound and Papillary Callose Formation.

Arabidopsis was transformed with double-stranded RNA interference (dsRNAi) constructs designed to silence three putative callose synthase genes: GLUCAN SYNTHASE-LIKE5 (GSL5), GSL6, and GSL11. Both wound callose and papillary callose were absent in lines transformed with GSL5 dsRNAi and in a corresponding sequence-indexed GSL5 T-DNA insertion line but were unaffected in GSL6 and GSL11 dsRNAi lines. These data provide strong genetic evidence that the GSL genes of higher plants encode proteins that are essential for callose formation. Deposition of callosic plugs, or papillae, at sites of fungal penetration is a widely recognized early response of host plants to microbial attack and has been implicated in impeding entry of the fungus. Depletion of callose from papillae in gsl5 plants marginally enhanced the penetration of the grass powdery mildew fungus Blumeria graminis on the nonhost Arabidopsis. Paradoxically, the absence of callose in papillae or haustorial complexes correlated with the effective growth cessation of several normally virulent powdery mildew species and of Peronospora parasitica.

Evidence that PSI-D, a chloroplast photosystem I protein, is in haustoria of the powdery mildew fungus Erysiphe pisi

Monoclonal antibody UB43 raised against isolated haustorial complexes (HC) of the pea powdery mildew fungus Erysiphe pisi, recognized an 18 kDa protein on Western blots of HC lysates but also of thylakoids isolated from pea chloroplasts. Microsequencing of the N-terminus of the antigen purified from chloroplasts, yielded a sequence of 13 amino acids with high homology to that of a photosystem I subunit (PSI-D). Co-identity of the UB43 antigen in chloroplasts and PSI-D, was confirmed by the use of authentic anti-PSI-D antibodies and immunoprecipitation procedures. These anti-PSI-D antibodies also bound to HCs and recognized an 18 kDa antigen. Immunofluorescence and EM-immunogold labelling showed that UB43 and the anti-PSI-D antibodies bound to the cytoplasm of haustorial lobes. Radiolabelled spinach PSI-D precursor produced by an in vitro transcription/translation system of PSI-D cDNA, was taken up by chloroplast-free HCs into a thermolysin-inaccessible compartment. PSI-D is located on the stromal side of the thylakoid membrane, and is thought to act as a docking site for the soluble ferredoxin. The nature and origin of the PSI-D related protein in HCs is discussed.

Components in the haustorial wall of the flax rust fungus,Melampsora lini, are labelled by three anti-calmodulin monoclonal antibodies

Immunolocalisation studies, using flax leaf material infected with the flax rust fungus,Melampsora lini, and isolated haustorial complexes, have shown that three anti-calmodulin monoclonal antibodies bind to the haustorial wall of the fungus. The epitopes recognised by these antibodies are inserted into the wall during the early stages of haustorium development and remain in the wall throughout the life of the haustorium. The epitopes are present in both compatible and incompatible reactions and are oligosaccharide in nature. The results provide evidence for molecular differentiation within the haustorial complex ofM. lini.

Morphology of haustoria of Ravenelia and Kernkampella spp.

The haustorial morphology of 21 Ravenelia species, two Kernkampella species, and four leguminicolous Uredo and Uraecium species which are probably related to Ravenelia, was investigated by light microscopy. Three different kinds of interaction could be found: (1) species with haustorial complexes comprising dikaryotic haustoria and intracellular hyphae. These species showed strictly intracellular growth of the parasitic mycelium. (2) Species with only dikaryotic haustoria, and (3) species with only intracellular hyphae or monokaryotic haustoria. Complexes of dikaryotic haustoria and intracellular hyphae were found in 17 of the investigated Ravenelia species and three Uredo species. Three Ravenelia species revealed dikaryotic haustoria, and Ravenelia lonchocarpicola had only intracellular hyphae. Complexes of dikaryotic haustoria and intracellular hyphae are probably the prevalent type of parasitic interaction in Ravenelia. The investigated Kernkampella species revealed dikaryotic haustoria.

A monoclonal antibody that recognizes a carbohydrate epitope on N-linked glycoproteins restricted to a subset of chitin-rich fungi

Previous studies with a monoclonal antibody (designated UB7) raised against isolated haustorial complexes formed by the pea powdery mildew fungus Erysiphe pisi showed that it recognized an abundant glycoprotein found in cell walls and plasma membranes of haustoria and the surface mycelium. In this paper, Western blotting and phase-partitioning in the detergent Triton X-114 have been used to show that the plasma membrane antigens recognized by UB7 comprise a 62 kDa integral glycoprotein and a set of integral membrane glycoproteins of lower molecular weight. A 59 kDa glycoprotein recognized by UB7 has been extracted from the fungus using aqueous buffer, and the evidence suggests that this represents the cell wall form of the antigen. Binding of UB7 is abolished by pre-treatment of glycoproteins with peptide-N-glycosidase, but is retained after endo-F treatment. This suggests that the epitope to which UB7 binds is the innermost N-acetylglucosamine residue of N-linked carbohydrate side chains of glycoproteins, possibly substituted with one or more sugars. When tested for cross-reactivity with other fungi, UB7 bound to some, but not all, of those examined, and recognized different sets of glycoproteins compared with those detected in E. pisi. However, UB7 did not bind to any higher plants nor to any animal glycoproteins tested. This antibody thus identifies a carbohydrate epitope restricted to glycoproteins in a subset of the fungi.

Molecular differentiation in the extrahaustorial membrane of pea powdery mildew haustoria at early and late stages of development

Two new monoclonal antibodies (MAbs) are described which recognise glycoprotein components of the extrahaustorial membrane ( ehm) which surrounds the haustorium formed during the infection of pea leaves by the powdery mildew fungus Erysiphe pisi. One of the MAbs, UB9, raised against enriched pea leaf plasma membranes, recognises a large (200 kDa) glycoprotein in this membrane within infected and uninfected epidermal cells. It does not label haustoria at early stages of development showing that at least one plasma membrane glycoprotein is excluded from the ehm. However, UB9 binds to the ehm of a subpopulation (approx 20%) of HCs late in the infection process and it is possible that the insertion of this glycoprotein into the ehm may represent a breakdown of biotrophy during senescence of haustoria or could be a late defence response by the plant. A second MAb, UB11, was raised against isolated haustorial complexes derived from the pea powdery mildew system. This antibody recognises a glycoprotein (250 kDa) which is present in the ehm of the haustorial complex and is expressed very early in haustorial development. UB11 does not bind to plant membranes of infected or uninfected plants and does not cross-react with other fungal components. This antibody therefore identifies a glycoprotein specifically located to the ehm of haustorial complexes and shows that there is molecular differentiation of this key interface between the biotrophic partners.

Glycoproteins recognised by monoclonal antibodies UB7, UB8 and UB10 are expressed early in the development of pea powdery mildew haustoria

A new monoclonal antibody (MAb) raised against haustorial complexes isolated from pea leaves infected with the powdery mildew fungus Erysiphe pisi is described. The antibody (designated UB10) recognises a 45 kDa N-linked glycoprotein which is specifically located in the haustorial plasma membrane. This glycoprotein is clearly distinct from a previously characterized 62 kDa glycoprotein (identified with MAb UB8) which is also specifically located in the haustorial plasma membrane. These antibodies were used, along with MAb UB7 which binds to a major 62 kDa glycoprotein in the cell wall and plasma membrane of both haustoria and surface hyphae, to label haustoria within epidermal strips from infected pea leaves using indirect immunofluorescence. Different stages of haustorial development can be observed using this procedure and the results showed that all three glycoproteins recognised by the MAbs are expressed early haustorial development.

Isolation of haustoria from wheat leaves infected by the leaf rust fungus

A procedure was developed to isolate haustoria from wheat leaves infected by Puccinia recondita . Homogenization of heavily infected leaves in an isolation medium was followed by a period of sedimentation. The resulting suspension was subjected to mild centrifugation and the pellet was separated further by centrifugation on a density gradient. One layer was selected and washed beforc examination by optical microscopy. Two types of fungal structure were distinguished by their shape, size and cytoplasmic activity and with a nuclear stain and lectin probes. One type was identified as hyphae and the other as haustoria. The reasons for identification are discussed in relation to literature on isolated haustorial complexes from powdery mildew fungi and on electron microscopic examination of sections of rust haustoria in wheat leaves.

Molecular differentiation in pea powdery-mildew haustoria

Monoclonal antibodies have been raised against haustorial complexes isolated from pea (Pisum sativum L.) leaves infected by the biotrophic powdery mildew fungus Erysiphe pisi D.C. Immuno-localisation studies, using isolated haustorial complexes and infected pea leaf material, have shown that one of the antibodies, designated UB7, binds to fungal wall and plasma membranes present in both haustoria and mycelia. However, a second antibody, UB8, binds specifically to the haustorial plasma membrane, and does not label fungal plasma membranes in mycelia. Western blotting and antigen-modification techniques have shown that UB8 recognises a protein epitope of a 62-kDa antigen. A reduction in molecular weight of this component after endo-F treatment indicates that the antigen is an N-linked glycoprotein. UB7 also recognises a 62-kDa glycoprotein, which is susceptible to endo-F treatment, and the antibody binds to a carbohydrate epitope. Differences in molecular weights of the products after endo-F treatment of antigens show that the 62-kDa glycoproteins recognised by the antibodies are distinct molecules, in accordance with the localisation results. Overall, the results provide evidence for molecular differentiation associated with the development of haustoria in a biotrophic infection.

The composition of Erysiphe pisi haustorial complexes with special reference to the neckbands

Haustorial complexes of Erysiphe pisi were isolated from the Pisum sativum, mutant line Argenteum, and their composition studied using fluorescent stains and enzymic digestions. Special attention was paid to the composition of the neckbands. Both A and B neckbands were stained with dyes specific for β-glucans, lipidic substances, protein and sulphydryl groups. The A band seemed to be richer in β-1,3-glucans than the B band, which more often contained β-1,4-glucans. The B band also contained proteinaceous compounds more often than the A band. Both neckbands were rich in lipidic substances. However, the organization of lipidic compounds and their attachment to other wall components differed in the A and B bands as indicated by digestion with lipase, esterase, zymolase, cellulase and Novozym 234, chitinase, ligninase, laccase and combinations of these enzymes. In the A band, lipidic components appeared to be more highly esterified than those in the B band, and bound to β-1,3-glucans and chitin. In the B band, attachment of the lipidic substances to a proteinaceous component was indicated, with masking of the lipids by wall polysaccharides. The junction between the extrahaustorial membrane and the haustorial neck at the neckbands was never seen to be completely degraded.

Permeability of the haustorium-host interface in powdery mildews

The extent to which uranyl ions and peroxidase permeated haustorial complexes isolated from Pisum sativum infected with Erysiphe pisi was determined by transmission electron microscopy (TEM). Freshly isolated complexes were treated with uranyl acetate, either directly, or after treatment with cell wall degrading enzymes, or with Triton X-100 and the uranyl ion, precipitated with phosphate. The complexes were then fixed with glutaraldehyde and examined by TEM to determine the distribution of uranyl phosphate crystals. Two independent experiments, each involving the examination of 80 complexes, gave similar results. After direct uranyl treatment, crystals were excluded from 38% of the complexes, were present in the extrahaustorial matrix only in 19% and occurred throughout in 43%. Breaks were observed in the extrahaustorial membrane of the last two categories. Uranyl crystals bound to the outer (host cytoplasmic) surface of the extrahaustorial membrane and also to the matrical surface, where this was accessible. In addition, the few necrotic haustorial complexes observed were completely permeated. Following pre-treatments with pectinase and cellulase, or Triton X-100, uranyl crystals were present throughout the complexes in all or 96%, of the complexes respectively. In similar experiments involving treatment with horseradish peroxidase (instead of uranyl acetate) followed by the addition of hydrogen peroxide and 3,3-diaminobenzidene, electron opaque deposit occurred around the complexes but never internally in any part of them. Even where breaks in the extrahaustorial membrane were visible the extrahaustorial matrix was not permeated. It is concluded that the extrahaustorial membrane and haustorial plasma membrane are semipermeable in many of the isolated haustorial complexes but that some are damaged during isolation and purification procedures; that a barrier to diffusion exists where the extrahaustorial membrane is joined to the wall of the haustorial neck and in other parts of the neck wall so that the whole boundary of the extrahaustorial matrix restricts diffusion of solutes; and that the extrahaustorial matrix is a gel which precludes the passage of large molecules through it. The physiological implications of the results are discussed.

TRANSPORT, TRANSLOCATION AND METABOLISM OF 14C‐PHOTOSYNTHATES AT THE HOST‐PARASITE INTERFACE OF PISUMSATIVUM AND ERYSIPHE PISI

SUMMARYHaustorial complex, mycelial and host (leaf without mycelia) samples were obtained from leaves of Pisum sativum infected with Erysiphe pisi either immediately following a 20 min exposure to 14CO2 (‘pulse’) or after a further 20 h ‘chase’ in 12CO2. After the ‘pulse’, labelled ethanol‐soluble sugars (glucose, fructose and sucrose) and mannitol predominated in the host and mycelial fractions respectively and lipids (mainly triglycerides) and an ethanol‐insoluble glucan (not α‐1, 4 linked) contained most of the 14C in the haustorial complexes. Ethanol‐soluble carbohydrates contained only 3% of the total 14C in the haustorial complexes with sucrose and glycerol as the major components. After the ‘chase’, the total 14C in host and mycelium declined but the proportions in starch or glycogen, protein, cell wall and nucleic acid fractions increased. Total 14C in haustorial complexes increased but that contained in sucrose and glycerol decreased and that in mannitol increased. During both ‘pulse’ and ‘chase’, the quantity of 14C in the haustorial complexes was small compared with that in the mycelial and leaf fractions.These results indicate that transport of host sugars to the fungus is mediated by haustoria. Sucrose and glycerol are either translocates or metabolic intermediates in this process and mannitol is the major primary sink of the fungus. Glycogen and other macromolecules are produced at a later stage in the fungus. Polysaccharides and lipids are important sinks at the host‐haustorial interface. These results are discussed in relation to the regulation of transport at the host‐parasite interface.

Accumulation of systemic fungicides and other solutes by haustorial complexes isolated from Pisum sativum infected with Erysiphe pisi

AbstractHaustorial complexes, isolated from pea leaves infected with powdery mildew, were incubated with individual 14C‐labelled systemic fungicides or other solutes, and the accumulation of these compounds by the complexes was assessed following filter centrifugation of the products through silicone oil. Inulincarboxylic acid and butyric acid were not absorbed; glucose, sucrose, acetic acid and glycerol were absorbed very slowly by the complexes. All the systemic fungicides, which included nuarimol, fenarimol, dimethirimol, ethirimol, and other pyrimidine‐based analogues, were accumulated against large concentration gradients (30‐1500 fold). The fatty acids, linoleic and lauric, were also readily accumulated. The accumulation of the fatty acids and fungicides was usually complete within 5–15 min. The degree of accumulation of all the solutes was significantly correlated with their lipophilicity, indicating that partitioning of compounds into sub‐cellular lipids was the major mechanism determining accumulation by the complexes. The cations 2‐hydroxyethylarnmonium and 1‐methylpyridinium were also accumulated against large concentration gradients (18‐and 46‐fold, respectively) but less rapidly than the fungicides. The significance of these findings for the systemic distribution and mode of action of fungicides is discussed.

Fluxes and accumulation of ethirimol in haustoria of Erysiphe pisi and protoplasts of Pisum sativum

SUMMARYIsolated purified fractions containing haustorial complexes and mesophyll protoplasts were used to investigate the relative affinities in vitro of the host‐parasite interface and host for the mildew specific fungicide, ethirimol. Compounds labelled with [14C] were used throughout this study.Isolated haustorial complexes accumulated fungicide against a concentration gradient and, in 15 min, to much higher concentrations than they accumulated glucose, sucrose, uracil, glycine, ethanolamine hydrochloride, inulin carboxylic acid, thiocyanate ions or uridine diphosphate glucose. Accumulation of ethirimol ceased after 30 min when the internal concentration was over twenty times greater than in the ambient medium. Similar quantities were absorbed in 15 min at pH 4.2 and 6–2. The quantities absorbed during 1 h incubation were directly related to the ambient concentration (4–400 μm). Ethirimol introduced into haustorial complexes was easily removed by washing; one third was removed in the first 5 min and only one sixth of the original remained after 3 h. Analysis of the kinetics of ethirimol efflux showed that it was located in two compartments within the complexes; thus it most probably entered the fungal cytoplasam.Ethirimol entered pea mesophyll protoplasts showing biphasic kinetics with considerable uptake in the first 30 min and a more gradual influx during the following 18 h. It was not accumulated against a concentration gradient until 6 h and after 18 h the internal concentration exceeded that of the ambient by only 1.74 fold.Extraction and chromatography showed that only small proportions of the ethirimol were degraded by haustorial complexes and protoplasts during the experimental periods.These in vitro experiments indicate competitive accumulation of ethirimol by the host‐parasite interface in vivo.

Electron microscope autoradiography of14C photosynthate distribution at the haustorium-host interface in powdery mildew ofPisum sativum

Haustorial complexes were isolated from leaves ofPisum sativum infected withErysiphe pisi and exposed to14CO2 for 2 hours. The constituents of the isolated fraction were quantified and ultrastructurally described and the distribution of14C studied by electron microscope autoradiography and statistical treatment. Most (86%) of the isotope in the fraction was associated with haustorial complexes. Three classes of haustorial complexes were distinguished by degree of labelling and ultrastructure. Most of the haustorial complexes were termed healthy (i.e., they showed a normal ultrastructure) and were heavily labelled; necrotic complexes were unlabelled; and a class with intermediate labelling, modified extrahaustorial membrane and usually a normal haustorial cytoplasm was termed pre-necrotic. In healthy haustorial complexes the haustorial lobes and extrahaustorial membrane showed the highest grain densities and the body and extrahaustorial matrix were also significantly labelled. Comparison of the results suggest that ultrastructural modifications leading to necrosis were caused by dehydration which in turn determined reduction in photosynthate transfer. Other factors influencing transport into haustoria and the status of the extrahaustorial matrix are also discussed.