Growth Inhibition of Phytopathogenic Fungi and Oomycetes by Basidiomycete Irpex lacteus and Identification of its Antimicrobial Extracellular Metabolites.

In dual culture confrontation assays, basidiomycete Irpex lacteus efficiently antagonized Fusarium spp., Colletotrichum spp., and Phytophthora spp. phytopathogenic strains, with growth inhibition percentages between 16.7-46.3%. Antibiosis assays evaluating the inhibitory effect of soluble extracellular metabolites indicated I. lacteus strain inhibited phytopathogens growth between 32.0-86.7%. Metabolites in the extracellular broth filtrate, identified by UPLC-QTOF mass spectrometer, included nine terpenes, two aldehydes, and derivatives of a polyketide, a quinazoline, and a xanthone, several of which had antifungal activity. I. lacteus strain and its extracellular metabolites might be valuable tools for phytopathogenic fungi and oomycete biocontrol of agricultural relevance.

A rust fungal effector binds plant DNA and modulates transcription.

The basidiomycete Melampsora larici-populina causes poplar rust disease by invading leaf tissues and secreting effector proteins through specialized infection structures known as haustoria. The mechanisms by which rust effectors promote pathogen virulence are poorly understood. The present study characterized Mlp124478, a candidate effector of M. larici-populina. We used the models Arabidopsis thaliana and Nicotiana benthamiana to investigate the function of Mlp124478 in plant cells. We established that Mlp124478 accumulates in the nucleus and nucleolus, however its nucleolar accumulation is not required to promote growth of the oomycete pathogen Hyaloperonospora arabidopsidis. Stable constitutive expression of Mlp124478 in A. thaliana repressed the expression of genes involved in immune responses, and also altered leaf morphology by increasing the waviness of rosette leaves. Chip-PCR experiments showed that Mlp124478 associats'e with the TGA1a-binding DNA sequence. Our results suggest that Mlp124478 exerts a virulence activity and binds the TGA1a promoter to suppress genes induced in response to pathogen infection.

Potential of aquatic oomycete as a novel feedstock for microbial oil grown on waste sugarcane bagasse.

Biodiesel production from vegetable oils is not sustainable and economical due to the food crisis worldwide. The development of a cost-effective non-edible feedstock is essential. In this study, we proposed to use aquatic oomycetes for microbial oils, which are cellulolytic fungus-like filamentous eukaryotic microorganisms, commonly known as water molds. They differ from true fungi as cellulose is present in their cell wall and chitin is absent. They show parasitic as well as saprophytic nature and have great potential to utilize decaying animal and plant debris in freshwater habitats. To study the triacylglycerol (TAG) accumulation in the aquatic oomycetes, the isolated water mold Achlya diffusa was cultivated under semi-solid-state conditions on waste sugarcane bagasse, which was compared with the cultivation in Czapek (DOX) medium. A. diffusa grown on waste sugarcane bagasse showed large lipid droplets in its cellular compartment and synthesized 124.03 ± 1.93 mg/gds cell dry weight with 50.26 ± 1.76% w/w lipid content. The cell dry weight and lipid content of this water mold decreased to 89.54 ± 1.21 mg/gds and 38.82% w/w, respectively, when cultivated on standard medium Czapek-Dox agar (CDA). For the fatty acid profile of A. diffusa grown in sugarcane bagasse and CDA, in situ transesterification (IST) and indirect transesterification (IDT) approaches were evaluated. The lipid profile of this mold revealed the presence of C12:0, C14:0, C16:0, C18:0, C18:1, C18:2, C20:0, and C21:0 fatty acids, which is similar to vegetable oils. The biodiesel properties of the lipids obtained from A. diffusa satisfied the limits as determined by international standards ASTM-D6751 and EN-14214 demonstrating its suitability as a fuel for diesel engines.

A new oomycete species parasitic in nematodes, Chlamydomyzium dictyuchoides sp. nov.: developmental biology and phylogenetic studies.

The genus Chlamydomyzium is a little studied holocarpic oomycete parasite of nematodes of uncertain phylogenetic and taxonomic position. A new holocarpic species, Chlamydomyzium dictyuchoides, is described which has usually refractile cytoplasm and a dictyuchoid pattern of spore release. This new species infects bacteriotrophic rhabditid nematodes and was isolated from diverse geographical locations. Infection was initiated by zoospore encystment on the host surface and direct penetration of the cuticle. A sparsely branched, constricted, refractile thallus was formed which eventually occupied almost the entire host body cavity, often accompanied by complete dissolution of the host cuticle. Walled primary cysts formed throughout the thallus and each cyst released a single zoospore via an individual exit papillum, leaving a characteristic dictyuchoid wall net behind. At later stages of infection some thalli formed thick-walled stellate resting spores in uniseriate rows. Resting spore formation appeared to be parthenogenetic and was not accompanied by the formation of antheridial compartments. These spores had ooplast-like vacuoles and thick multi-layered walls, both of which suggest they were oospores. The maximum likelihood tree of sequences of the small ribosomal subunit (SSU) gene placed this new isolate in a clade before the main saprolegnialean and peronosporalean lines diverge. A second undescribed Chlamydomyzium sp., which has direct spore release forms a paraphyletic clade, close to C. dictyuchoides and Sapromyces. The fine structure of other documented Chlamydomyzium species was compared, including an undescribed (but sequenced) isolate, SL02, from Japan, Chlamydomyzium anomalum and Chlamydomyzium oviparasiticum. Chlamydomyzium as currently constituted is a paraphyletic genus that is part of a group of phylogenetically problematic early diverging clades that lie close to both the Leptomitales and Rhipidiales.

Activity profiling of vacuolar processing enzymes reveals a role for VPE during oomycete infection.

Vacuolar processing enzymes (VPEs) are important cysteine proteases that are implicated in the maturation of seed storage proteins, and programmed cell death during plant-microbe interactions and development. Here, we introduce a specific, cell-permeable, activity-based probe for VPEs. This probe is highly specific for all four Arabidopsis VPEs, and labeling is activity-dependent, as illustrated by sensitivity for inhibitors, pH and reducing agents. We show that the probe can be used for in vivo imaging and displays multiple active isoforms of VPEs in various tissues and in both monocot and dicot plant species. Thus, VPE activity profiling is a robust, simple and powerful tool for plant research for a wide range of applications. Using VPE activity profiling, we discovered that VPE activity is increased during infection with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa). The enhanced VPE activity is host-derived and EDS1-independent. Sporulation of Hpa is reduced on vpe mutant plants, demonstrating a role for VPE during compatible interactions that is presumably independent of programmed cell death. Our data indicate that, as an obligate biotroph, Hpa takes advantage of increased VPE activity in the host, e.g. to mediate protein turnover and nutrient release.

Patterns of plant subcellular responses to successful oomycete infections reveal differences in host cell reprogramming and endocytic trafficking.

Adapted filamentous pathogens such as the oomycetes Hyaloperonospora arabidopsidis (Hpa) and Phytophthora infestans (Pi) project specialized hyphae, the haustoria, inside living host cells for the suppression of host defence and acquisition of nutrients. Accommodation of haustoria requires reorganization of the host cell and the biogenesis of a novel host cell membrane, the extrahaustorial membrane (EHM), which envelops the haustorium separating the host cell from the pathogen. Here, we applied live-cell imaging of fluorescent-tagged proteins labelling a variety of membrane compartments and investigated the subcellular changes associated with accommodating oomycete haustoria in Arabidopsis and N. benthamiana. Plasma membrane-resident proteins differentially localized to the EHM. Likewise, secretory vesicles and endosomal compartments surrounded Hpa and Pi haustoria revealing differences between these two oomycetes, and suggesting a role for vesicle trafficking pathways for the pathogen-controlled biogenesis of the EHM. The latter is supported by enhanced susceptibility of mutants in endosome-mediated trafficking regulators. These observations point at host subcellular defences and specialization of the EHM in a pathogen-specific manner. Defence-associated haustorial encasements, a double-layered membrane that grows around mature haustoria, were frequently observed in Hpa interactions. Intriguingly, all tested plant proteins accumulated at Hpa haustorial encasements suggesting the general recruitment of default vesicle trafficking pathways to defend pathogen access. Altogether, our results show common requirements of subcellular changes associated with oomycete biotrophy, and highlight differences between two oomycete pathogens in reprogramming host cell vesicle trafficking for haustoria accommodation. This provides a framework for further dissection of the pathogen-triggered reprogramming of host subcellular changes.

Subcellular localization of the Hpa RxLR effector repertoire identifies a tonoplast-associated protein HaRxL17 that confers enhanced plant susceptibility.

Filamentous phytopathogens form sophisticated intracellular feeding structures called haustoria in plant cells. Pathogen effectors are likely to play a role in the establishment and maintenance of haustoria in addition to their better-characterized role in suppressing plant defence. However, the specific mechanisms by which these effectors promote virulence remain unclear. To address this question, we examined changes in subcellular architecture using live-cell imaging during the compatible interaction between the oomycete Hyaloperonospora arabidopsidis (Hpa) and its host Arabidopsis. We monitored host-cell restructuring of subcellular compartments within plant mesophyll cells during haustoria ontogenesis. Live-cell imaging highlighted rearrangements in plant cell membranes upon infection, in particular to the tonoplast, which was located close to the extra-haustorial membrane surrounding the haustorium. We also investigated the subcellular localization patterns of Hpa RxLR effector candidates (HaRxLs) in planta. We identified two major classes of HaRxL effector based on localization: nuclear-localized effectors and membrane-localized effectors. Further, we identified a single effector, HaRxL17, that associated with the tonoplast in uninfected cells and with membranes around haustoria, probably the extra-haustorial membrane, in infected cells. Functional analysis of selected effector candidates in planta revealed that HaRxL17 enhances plant susceptibility. The roles of subcellular changes and effector localization, with specific reference to the potential role of HaRxL17 in plant cell membrane trafficking, are discussed with respect to Hpa virulence.

Changes in carbohydrate metabolism in Plasmopara viticola-infected grapevine leaves.

The oomycete Plasmopara viticola is responsible for downy mildew, a severe grapevine disease. In infected grapevine leaves, we have observed an abnormal starch accumulation at the end of the dark period, suggesting modifications in starch metabolism. Therefore, several complementary approaches, including transcriptomic analyses, measurements of enzyme activities, and sugar quantification, were performed in order to investigate and to understand the effects of P. viticola infection on leaf starch and-to a larger extent-carbohydrate metabolism. Our results indicate that starch accumulation is associated with an increase in ADP-glucose pyrophosphorylase (AGPase) activity and modifications in the starch degradation pathway, especially an increased α-amylase activity. Together with these alterations in starch metabolism, we have observed an accumulation of hexoses, an increase in invertase activity, and a reduction of photosynthesis, indicating a source-to-sink transition in infected leaf tissue. Additionally, we have measured an accumulation of the disaccharide trehalose correlated to an increased trehalase gene expression and enzyme activity. Altogether, these results highlight a dramatic alteration of carbohydrate metabolism correlated with later stages of P. viticola development in leaves.

The development, ultrastructural cytology, and molecular phylogeny of the basal oomycete Eurychasma dicksonii, infecting the filamentous phaeophyte algae Ectocarpus siliculosus and Pylaiella littoralis.

The morphological development, ultrastructural cytology, and molecular phylogeny of Eurychasma dicksonii, a holocarpic oomycete endoparasite of phaeophyte algae, were investigated in laboratory cultures. Infection of the host algae by E. dicksonii is initiated by an adhesorium-like infection apparatus. First non-walled, the parasite cell developed a cell wall and numerous large vacuoles once it had almost completely filled the infected host cell (foamy stage). Large-scale cytoplasmic changes led to the differentiation of a sporangium with peripheral primary cysts. Secondary zoospores appeared to be liberated from the primary cysts in the internal space left after the peripheral spores differentiated. These zoospores contained two phases of peripheral vesicles, most likely homologous to the dorsal encystment vesicles and K-bodies observed in other oomycetes. Following zoospore liberation the walls of the empty cyst were left behind, forming the so-called net sporangium, a distinctive morphological feature of this genus. The morphological and ultrastructural features of Eurychasma were discussed in relation to similarities with other oomycetes. Both SSU rRNA and COII trees pointed to a basal position of Eurychasma among the Oomycetes. The cox2 sequences also revealed that the UGA codon encoded tryptophan, constituting the first report of stop codon reassignment in an oomycete mitochondrion.

A beta-1,3 glucan sulfate induces resistance in grapevine against Plasmopara viticola through priming of defense responses, including HR-like cell death.

Sulfated laminarin (PS3) has been shown previously to be an elicitor of plant defense reactions in tobacco and Arabidopsis and to induce protection against tobacco mosaic virus. Here, we have demonstrated the efficiency of PS3 in protecting a susceptible grapevine cultivar (Vitis vinifera cv. Marselan) against downy mildew (Plasmopara viticola) under glasshouse conditions. This induced resistance was associated with potentiated H2O2 production at the infection sites, upregulation of defense-related genes, callose and phenol depositions, and hypersensitive response-like cell death. Interestingly, similar responses were observed following P. viticola inoculation in a tolerant grapevine hybrid cultivar (Solaris). A pharmacological approach led us to conclude that both callose synthesis and jasmonic acid pathway contribute to PS3-induced resistance.

The drought response of Theobroma cacao (cacao) and the regulation of genes involved in polyamine biosynthesis by drought and other stresses.

Drought can negatively impact pod production despite the fact that cacao production usually occurs in tropical areas having high rainfall. Polyamines (PAs) have been associated with the response of plants to drought in addition to their roles in responses to many other stresses. The constitutive and drought inducible expression patterns of genes encoding enzymes involved in PA biosynthesis were determined: an ornithine decarboxylase (TcODC), an arginine decarboxylase (TcADC), an S-adenosylmethionine decarboxylase (TcSAMDC), a spermidine synthase (TcSPDS), and a spermine synthase (TcSPMS). Expression analysis using quantitative real-time reverse transcription-PCR (QPCR) results showed that the PA biosynthesis genes were expressed in all plant tissues examined. Constitutive expression of PA biosynthesis genes was generally highest in mature leaves and open flowers. Expression of TcODC, TcADC, and TcSAMDC was induced with the onset of drought and correlated with changes in stomatal conductance, photosynthesis, photosystem II efficiency, leaf water potential and altered emission of blue-green fluorescence from cacao leaves. Induction of TcSAMDC in leaves was most closely correlated with changes in water potential. The earliest measured responses to drought were enhanced expression of TcADC and TcSAMDC in roots along with decreases in stomatal conductance, photosynthesis, and photosystem II efficiency. Elevated levels of putrescine, spermidine, and spermine were detected in cacao leaves 13days after the onset of drought. Expression of all five PA associated transcripts was enhanced (1.5-3-fold) in response to treatment with abscisic acid. TcODC and TcADC, were also responsive to mechanical wounding, infection by Phytophthora megakarya (a causal agent of black pod disease in cacao), the necrosis- and ethylene-inducing protein (Nep1) of Fusarium oxysporum, and flower abscission. TcSAMDC expression was responsive to all stresses except flower abscission. TcODC, although constitutively expressed at much lower levels than TcADC, TcSAMDC, TcSPDS, and TcSPMS, was highly inducible by the fungal protein Nep1 (135-fold) and the cacao pathogen Phytophthora megakarya (671-fold). The full length cDNA for ODC was cloned and characterized. Among the genes studied, TcODC, TcADC, and TcSAMDC were most sensitive to induction by drought in addition to other abiotic and biotic stresses. TcODC, TcADC, and TcSAMDC may share signal transduction pathways and/or the stress induced signal induction pathways may converge at these three genes leading to similar although not identical patterns of expression. It is possible altering PA levels in cacao will result in enhanced tolerance to multiple stresses including drought and disease as has been demonstrated in other crops.

An ultrastructural study of development and reproduction in the nematode parasite Myzocytiopsis vermicola.

An isolate of Myzocytiopsis vermicola, a holocarpic parasite of Rhabditis nematodes, was studied with transmission electron microscopy (TEM) to follow development during infection, asexual and sexual reproduction. Nematodes became infected after attachment of apical cystospore buds to the nematode cuticle. Apical buds were packed with vesicles with dense fibrillar contents, which were absent from the thallus. Some thalli developed into sporangia while others became paired gametangial cells. Zoospore cleavage was often intrasporangial, although during the early stages of an epidemic partially differentiated zoospores usually were released via an exit tube into a fine vesicle. Packets of tripartite tubular hairs (TTH) were not observed in the cytoplasm of either developing or mature sporangia. TEM of sectioned material and whole mounts of zoospores revealed biflagellate zoospores, some without hairs and others with a proximal row of very short hairs on the anterior flagellum. Gametangial contact was via a short, walled fertilization tube and surplus antheridial and oogonial nuclei remained in their respective gametangial cells until disintegration of the periplasm. The mature oospores had a scalloped, electron opaque, epispore wall layer. These observations will be discussed in relation to the likely phylogenetic position of the Myzocytiopsidales within the oomycetes.

Coordinated regulation of genes for secretion in tobacco at late developmental stages: association with resistance against oomycetes.

Besides the systemic acquired resistance (SAR) induced in response to microbial stimulation, host plants may also acquire resistance to pathogens in response to endogenous stimuli associated with their own development. In tobacco (Nicotiana tabacum), the vegetative-to-flowering transition comes along with a susceptibility-to-resistance transition to the causal agent of black shank disease, the oomycete Phytophthora parasitica. This resistance affects infection effectiveness and hyphal expansion and is associated with extracellular accumulation of a cytotoxic activity that provokes in vitro cell death of P. parasitica zoospores. As a strategy to determine the extracellular events important for restriction of pathogen growth, we screened the tobacco genome for genes encoding secreted or membrane-bound proteins expressed in leaves of flowering plants. Using a signal sequence trap approach in yeast (Saccharomyces cerevisiae), 298 clones were selected that appear to encode for apoplastic, cell wall, or membrane-bound proteins involved in stress response, in plant defense, or in cell wall modifications. Microarray and northern-blot analyses revealed that, at late developmental stages, leaves were characterized by the coordinate up-regulation of genes involved in SAR and in peroxidative cross-linking of structural proteins to cell wall. This suggests the potential involvement of these genes in extracellular events that govern the expression of developmental resistance. The analysis of the influence of salicylic acid on mRNA accumulation also indicates a more complex network for regulation of gene expression at a later stage of tobacco development than during SAR. Further characterization of these genes will permit the formulation of hypotheses to explain resistance and to establish the connection with development.

Antagonistic activity of Aeromonas media strain A199 against Saprolegnia sp., an opportunistic pathogen of the eel, Anguilla australis Richardson.

A bacteriocin-like inhibitory substance (BLIS) produced by Aeromonas media strain A199 inhibited the growth of Saprolegnia sp. in vitro, an opportunistic pathogen isolated from affected eels, Anguilla australis (Richardson). The presence of BLIS in solid media inhibited the growth of the vegetative state of the aquatic mould as well as the germination of cysts. Uninhibited growth was, however, observed in the presence of inactive BLIS, suggesting that the in vitro antagonism derived from the BLIS of A199. In four independent in vivo tank observations of fish affected with saprolegniosis, the daily addition of A199 to tank water contributed to the subsequent swift recovery of affected hosts from invasion by this opportunistic pathogen.

Ectopic expression of Tsi1 in transgenic hot pepper plants enhances host resistance to viral, bacterial, and oomycete pathogens.

In many plants, including hot pepper plants, productivity is greatly affected by pathogen attack. We reported previously that tobacco stress-induced gene 1 (Tsi1) may play an important role in regulating stress responsive genes and pathogenesis-related (PR) genes. In this study, we demonstrated that overexpression of Tsi1 gene in transgenic hot pepper plants induced constitutive expression of several PR genes in the absence of stress or pathogen treatment. The transgenic hot pepper plants expressing Tsi1 exhibited resistance to Pepper mild mottle virus (PMMV) and Cucumber mosaic virus (CMV). Furthermore, these transgenic plants showed increased resistance to a bacterial pathogen, Xanthomonas campestris pv. vesicatoria and also an oomycete pathogen, Phytophthora capsici. These results suggested that ectopic expression of Tsi1 in transgenic hot pepper plants enhanced the resistance of the plants to various pathogens, including viruses, bacteria, and oomycete. These results suggest that using transcriptional regulatory protein genes may contribute to developing broad-spectrum resistance in crop plants.

Pumilio homologue from saprolegnia parasitica specifically expressed in undifferentiated spore cysts.

The expression of spore-specific marker transcripts at different stages of the asexual life cycle of Saprolegnia parasitica was analyzed. One of the markers, designated puf1, was found to be expressed transiently upon each of several cycles of zoospore encystment and reemergence. The transcript is induced immediately upon zoospore encystment and is rapidly lost when a cyst is triggered to germinate. In nongerminating cysts, puf1 is maintained until a time point when the cysts can no longer be triggered to germinate and thus have become determined for zoospore reemergence. The results show that the cyst stage has two phases, of about equal duration, which are physiologically and transcriptionally distinct and that the transcriptional machinery of oomycetes is also active in nongerminating spores. puf1 encodes a putative mRNA binding protein belonging to a conserved class of proteins including the Drosophila melanogaster Pumilio protein, Caenorhabditis elegans FBF, and Saccharomyces cerevisiae Puf5, all of which are involved in regulation of gene expression by post-transcriptional mechanisms.

From elicitins to lipid-transfer proteins: a new insight in cell signalling involved in plant defence mechanisms.

Elicitins and lipid-transfer proteins are small cysteine-rich lipid-binding proteins secreted by oomycetes and plant cells, respectively, that share some structural and functional properties. In spite of intensive work on their structure and diversity at the protein and genetic levels, the precise biological roles of lipid-transfer proteins remains unclear, although the most recent data suggest a role in somatic embryogenesis, in the formation of protective surface layers and in defence against pathogens. By contrast, elicitins are known elicitors of plant defence, and recent work demonstrating that elicitins and lipid-transfer proteins share the same biological receptors gives a new perspective to understand the role played by lipid binding proteins, mainly the early recognition of intruders in plants.

The Arabidopsis hrl1 mutation reveals novel overlapping roles for salicylic acid, jasmonic acid and ethylene signalling in cell death and defence against pathogens.

Defence against pathogens in Arabidopsis is orchestrated by at least three signalling molecules: salicylic acid (SA), jasmonic acid (JA) and ethylene (ET). The hrl1 (hypersensitive response-like lesions 1) mutant of Arabidopsis is characterized by spontaneous necrotic lesions, accumulation of reactive oxygen species, constitutive expression of SA- and ET/JA-responsive defence genes, and enhanced resistance to virulent bacterial and oomycete pathogens. Epistasis analyses of hrl1 with npr1, etr1, coi1 and SA-depleted nahG plants revealed novel interactions between SA and ET/JA signalling pathways in regulating defence gene expression and cell death. RNA gel-blot analysis of RNA isolated separately from the lesion+ and the lesion- leaves of double mutants of hrl1 revealed different signalling requirements for the expression of defence genes in these tissues. Expression of the ET/JA-responsive PDF1.2 gene was markedly reduced in hrl1 npr1 and in SA-depleted hrl1 nahG plants. In hrl1 nahG plants, expression of PDF1.2 was regulated by benzathiadiazole in a concentration-dependent manner: induced at low concentration and suppressed at high concentration. The hrl1 etr1 plants lacked systemic PR-1 expression, and exhibited compromised resistance to virulent Pseudomonas syringae and Peronospora parasitica. Inhibiting JA responses in hrl1 coi1 plants lead to exaggerated cell death and severe stunting of plants. Finally, the hrl1 mutation lead to elevated expression of AtrbohD, which encodes a major subunit of the NADPH oxidase complex. Our results indicate that defence gene expression and resistance against pathogens in hrl1 is regulated synergistically by SA and ET/JA defence pathways.

A tobacco S-like RNase inhibits hyphal elongation of plant pathogens.

Ribonuclease (RNase) NE gene expression is induced in tobacco leaves in response to Phytophthora parasitica. Using antibodies directed against RNase NE, we demonstrate that RNase NE is extracellular at the early steps of the interaction, while the fungal tip growth is initiated in the apoplastic compartment. After production in Pichia pastoris and biochemical purification, we show that the S-like RNase NE inhibits hyphal growth from P. parasitica zoospores and from Fusarium oxysporum conidia in vitro. Conversion into an enzymatically inactive form after mutagenesis of the active site-histidine 97 residue to phenylalanine leads to the suppression of this activity, suggesting that RNase NE inhibits the elongation of germ tubes by degradation of microbial RNAs. Exogenous application of RNase NE in the extracellular space of leaves inhibits the development of P. parasitica. Based on its induction by inoculation, its localization, and its activity against two plant pathogens, we propose that RNase NE participates in tobacco defense mechanisms by a direct action on hyphal development in the extracellular space. The RNase activity-dependent antimicrobial activity of the S-like RNase NE shares similarities with the only other biological activity demonstrated for plant RNases, the inhibition of elongation of pollen tubes by the S-RNase in gametophytic self-incompatibility, suggesting a functional link between self and nonself interactions in plants.

Proteolytic release of membrane-bound endo-(1,4)-beta-glucanase activity associated with cell wall softening in Achlya ambisexualis.

Branching and other cell wall softening events in fungi and oomycetes are thought to involve the activity of secreted enzymes, which are packaged in membrane vesicles and delivered to sites of cell expansion, there to work in a carefully regulated manner upon the structure of the wall. Here we demonstrate a latent endo-(1,4)-beta-glucanase activity in a mixed membrane fraction of the oomycete Achlya ambisexualis, which can be released by cysteine proteases with an increase of apparent activity. In addition, a similar endogenous process is strongly inhibited by the cysteine protease inhibitor iodoacetamide, while inhibitors of other types of proteases have a much smaller effect. Detergent treatment of membranes releases two glucanases detectable by electrophoretic activity staining, with apparent molecular masses of about 164 and 35 kDa. Proteolysis produces several activity bands, with major species having apparent molecular masses of about 149, 133, 48, 35, and 25 kDa. The ca. 35- and 25-kDa bands migrate in parallel with glucanases secreted during wall softening in vivo. We propose that the initiation of wall softening in Achlya involves the proteolytic processing and solubilization of at least some secreted endoglucanases. We also propose that the solubilization component of this process functions not just to provide the enzymes with access to wall matrix substrates but also may provide a mechanism for the eventual termination of their biological function.