First report of Achlya bisexualis infection in captive-reared Endangered golden mahseer Tor putitora.

Achlya bisexualis is a notorious oomycete pathogen with the potential to cause emerging disease in fish farms. In this study, we report the first isolation of A. bisexualis from captive-reared golden mahseer Tor putitora, an Endangered fish species. The infected fish showed a cotton-like growth of mycelia at the site of infection. The mycelium when cultured on potato dextrose agar produced radially growing white hyphae. The hyphae were non-septate, and some of them carried matured zoosporangium with dense granular cytoplasmic contents. Spherical gemmae with stout stalks were also observed. All the isolates had 100% identity in internal transcribed spacer (ITS)-rDNA sequence and showed highest similarity to that of A. bisexualis. In molecular phylogeny, all the isolates formed a monophyletic group with A. bisexualis which was supported by a bootstrap value of 99%. Based on the molecular and morphological findings, all the isolates were confirmed as A. bisexualis. Further, the anti-oomycete effect of boric acid, a known antifungal agent, against the isolate was evaluated. The minimum inhibitory concentration and minimum fungicidal concentration were found to be 1.25 and >2.5 g l-1, respectively. Isolation of A. bisexualis from a new fish species indicates its possible occurrence in other unreported hosts. Considering its wide infectivity and the potential to cause disease in farmed fishes, its probable prevalence in a new environment and host needs to be closely monitored to prevent the spread of infection, if any, by adopting suitable control measures.

Biomechanical responses of encysted zoospores of the oomycete Achlya bisexualis to hyperosmotic stress are consistent with an ability to turgor regulate.

Zoospores are motile, asexual reproductive propagules that enable oomycete pathogens to locate and infect new host tissue. While motile, they have no cell wall and maintain tonicity with their external media using water expulsion vacuoles. Once they locate host tissue, they encyst and form a cell wall, enabling the generation of turgor pressure that will provide the driving force for germination and invasion of the host. It is not currently known how these spores respond to the osmotic stresses that might arise due to different environments on and around their hosts that have different osmotic strengths. We have made microaspiration (MA) measurements on > 800 encysted zoospores and atomic force microscopy (AFM) measurements on 12 encysted zoospores to determine their mechanical properties and how these change after hyperosmotic stress. Two types of encysted zoospores (Type A and Type B) were produced from the oomycete Achlya bisexualis, that differed in their morphology and response. With a small hyperosmotic stress (using 0.1 and 0.2 M sorbitol to give media osmolality changes of 155.4 and 295.6 mOsmol/kg), Type A zoospores initially became stiffer, with an increase in the Young's modulus (E) over 30 mins from 0.16 MPa to 0.25 and 0.22 MPa respectively. E then returned to its original value after 120 min. With a greater osmotic stress (using 0.3, 0.4 and 0.5 M sorbitol to give media osmolality changes of 438.2, 587.2 and 787.6 mOsmol/kg) the reverse occurred, with an initial decrease in E over 30 - 60 mins to values of 0.1, 0.08 and 0.09 MPa respectively, before recovery to the original value after 120 min. In 0.5 M sorbitol this recovery was only observed with AFM, but not with MA. Type B zoospores, which may be primary/secondary spores about to release secondary/tertiary spores, or else spores that were damaged during encystment, initially stiffened in response to the lower hyperosmotic stresses with a slight increase in E (from 0.077 to 0.1 MPa after 15 min (with both 0.1 and 0.2 M sorbitol) before recovering to the original value after 60 min. These spores showed no change in response to the higher osmotic stresses. The responses of the Type A spores are consistent with rapid changes in cell wall thickness and a turgor regulation mechanism. Turgor regulation is further supported by microscopic observations of the Type A spores showing protoplast retraction from the cell wall followed by deplasmolysis, coupled with measurements of spore volume. As far as we are aware this is the first demonstration of turgor regulation, not just in encysted zoospores, but in oomycetes in general.

Microfluidic platform for integrated compartmentalization of single zoospores, germination and measurement of protrusive force generated by germ tubes.

This paper describes the design, fabrication and characterisation of a novel monolithic lab-on-a-chip (LOC) platform combining the trapping and germination of individual zoospores of the oomycete Achlya bisexualis with elastomeric micropillar-based protrusive force sensing. The oomycetes are of significant interest due to their pathogenic capabilities, which can have profound ecological and economic impacts. Zoospore encystment and germination via a germ tube play a key role in their pathogenicity. Our platform enables the study of these processes at a single cell level through hydrodynamic trapping of zoospores and their individual compartmentalization via normally closed pneumatic membrane microvalves. Valve geometry was optimized and media exchange characterized during dynamic valve operations to enhance the capture-to-growth ratio. We demonstrate germination of A. bisexualis zoospores on the platform and report three distinct germination patterns. Once germinated, germ tubes grew down growth channels towards single elastomeric micropillars. Tracking of pillar movement allowed for the measurement of microNewton range protrusive forces imparted by the tips of the germ tubes. Results indicate that the forces generated by the germ tubes are smaller than those exerted by mature hyphae. Through the use of parallel traps, channels and pillars on the same device, the platform enables high-throughput screening (HTS) of zoospores and their generation of protrusive force, an essential component of their infective capability. Due to its versatility, it will also allow for the screening of naturally bioactive compounds and the development of new biocontrol strategies for oomycetes, and morphologically similar fungal infections, as an alternative to agrochemicals.

Achlya sp. Dermatitis in an American Alligator (Alligator mississippiensis).

Oomycetes are water moulds in the kingdom Protista and are not considered true fungi due to the structural lack of chitin and ergosterol. Many oomycetes are pathogenic, such as Pythium spp., and many fish are prone to Saprolegnia spp. infections, particularly in stressful farming situations. A juvenile American alligator (Alligator mississippiensis) was presented for necropsy examination with white, gelatinous, raised lesions over ulcerated regions of skin on the limbs and tail. The alligator came from a hatchery with age-divided enclosures, and several of the animals within the same enclosure showed similar lesions. Numerous hyphae with non-parallel walls and sparse, non-dichotomous branching were observed histologically on Gomori's methenamine silver staining within the ulcers. Although no organisms were detectable via polymerase chain reaction testing of fresh or formalin-fixed tissues, the organism was cultured and sequenced as an Achlya sp., an infrequently identified oomycete. To the author's knowledge, this is the first description of an oomycete infection within the class Reptilia.

First Report of Achlya oblongata Infection in Freshwater-Reared Asian Seabass Lates calcarifer.

In September 2014, a freshwater oomycete was first isolated from Asian Seabass Lates calcarifer fry that were reared in freshwater at a fish hatchery in Sabah, Malaysia. A fungal strain was isolated from infected fry by using glucose yeast extract (GY) agar. From morphological identification, the strain belonged to the genus Achlya based on the mode of zoospore release. Molecular phylogenetic analysis of the internal transcribed spacer region sequences from the strain showed high similarity (99-100%) to Achlya oblongata. The isolate was able to grow on GY agar incubated at 15-35°C, in GY broth adjusted to pH 3.0-11.0, and in up to 1.0% NaCl. This is the first report of Achlya infection in freshwater-reared Asian Seabass in Malaysia.

An elastomeric micropillar platform for the study of protrusive forces in hyphal invasion.

Oomycetes and fungi are microorganisms whose pathogenic (invasive) growth can cause diseases that are responsible for significant ecological and economic losses. Such growth requires the generation of a protrusive force, the magnitude and direction of which involves a balance between turgor pressure and localised yielding of the cell wall and the cytoskeleton. To study invasive growth in individual hyphae we have developed a lab-on-a-chip platform with integrated force-sensors based on elastomeric polydimethylsiloxane (PDMS) micro-pillars. With this platform we are able to measure protrusive force (both magnitude and direction) and hyphal morphology. To show the usefulness of the platform, the oomycete Achlya bisexualis was inoculated and grown on a chip. Growth of individual hyphae into a micro-pillar revealed a maximum total force of 10 µN at the hyphal tip. The chips had no discernible effect on hyphal growth rates, but hyphae were slightly thinner in the channels on the chips compared to those on agar plates. When the hyphae contacted the pillars tip extension decreased while tip width increased. A. bisexualis hyphae were observed to reorient their growth direction if they were not able to bend and effectively grow over the pillars. Estimates of the pressure exerted on a pillar were 0.09 MPa, which given earlier measures of turgor of 0.65 MPa would indicate low compliance of the cell wall. The platform is adaptable to numerous cells and organisms that exhibit tip-growth. It provides a useful tool to begin to unravel the molecular mechanisms that underlie the generation of a protrusive force.

A pressure gradient facilitates mass flow in the oomycete Achlya bisexualis.

We have used a single cell pressure probe and observed movement of microinjected oil droplets to investigate mass flow in the oomycete Achlya bisexualis. To facilitate these experiments, split Petri dishes that had media containing different sorbitol concentrations (and hence a different osmotic potential) on each side of the dish were inoculated with a single zoospore. An initial germ tube grew out from this and formed a mycelium that extended over both sides of the Petri dish. Hyphae growing on the 0 M sorbitol side of the dish had a mean turgor ( ± sem) of 0.53 ± 0.03 MPa (n = 13) and on the 0.3 M sorbitol side had a mean turgor ( ± sem) of 0.3 ± 0.027 MPa (n = 9). Oil droplets that had been microinjected into the hyphae moved towards the lower turgor area of the mycelia (i.e. retrograde movement when microinjected into hyphae on the 0 M sorbitol side of the split Petri dish and anterograde movement when microinjected into hyphae on the 0.3 M sorbitol side of the Petri dish). In contrast, the movement of small refractile vesicles occurred in both directions irrespective of the pressure gradient. Experiments with neutral red indicate that the dye is able to move through the mycelia from one side of a split Petri dish to the other, suggesting that there is no compartmentation. This study shows that hyphae that are part of the same mycelia can have different turgor pressures and that this pressure gradient can drive mass flow.

The secreted proteins of Achlya hypogyna and Thraustotheca clavata identify the ancestral oomycete secretome and reveal gene acquisitions by horizontal gene transfer.

Saprotrophic and parasitic microorganisms secrete proteins into the environment to breakdown macromolecules and obtain nutrients. The molecules secreted are collectively termed the "secretome" and the composition and function of this set of proteins varies depending on the ecology, life cycle, and environment of an organism. Beyond the function of nutrient acquisition, parasitic lineages must also secrete molecules to manipulate their host. Here, we use a combination of de novo genome and transcriptome sequencing and bioinformatic identification of signal peptides to identify the putative secreted proteome of two oomycetes, the facultative parasite Achlya hypogyna and free-living Thraustotheca clavata. By comparing the secretomes of these saprolegnialean oomycetes with that of eight other oomycetes, we were able to characterize the evolution of this protein set across the oomycete clade. These species span the last common ancestor of the two major oomycete families allowing us to identify the ancestral secretome. This putative ancestral secretome consists of at least 84 gene families. Only 11 of these gene families are conserved across all 10 secretomes analyzed and the two major branches in the oomycete radiation. Notably, we have identified expressed elicitin-like effector genes in the saprotrophic decomposer, T. clavata. Phylogenetic analyses show six novel horizontal gene transfers to the oomycete secretome from bacterial and fungal donor lineages, four of which are specific to the Saprolegnialeans. Comparisons between free-living and pathogenic taxa highlight the functional changes of oomycete secretomes associated with shifts in lifestyle.

An investigation into plasmolysis in the oomycete Achlya bisexualis reveals that membrane-wall attachment points are sensitive to peptides containing the sequence RGD and that cell wall deposition can occur despite retraction of the protoplast.

The structure and function of membrane-wall attachment sites in walled cells, and how these relate to animal focal adhesions, is an area that is poorly understood. In view of this, we investigated how membrane-wall attachments that form upon plasmolysis, respond to peptides that disrupt animal focal adhesions. The degree of cytoplasmic disruption during plasmolysis was also investigated. Upon hyperosmotic challenge, the protoplast in hyphae of the oomycete Achlya bisexualis typically retracted incompletely due to membrane-wall attachments. The inclusion, in the plasmolysing solution, of peptides containing the sequence RGD disrupted these attachments in a dose-dependent manner. In some hyphae, protoplast retraction stopped temporarily at attachment points - upon resumption of retraction, material was left that traced the outline of the static protoplast. Staining of this material with fluorescence brightener indicated the presence of cellulose, which suggests that wall deposition was able to occur despite plasmolysis. The F-actin cytoskeleton was disrupted during plasmolysis; peripheral F-actin staining was observed, but there was no distinct F-actin cap; staining was more diffuse; and there were fewer plaques compared with nonplasmolysed hyphae. Our data indicate that membrane-wall attachment points are sensitive to RGD-containing peptides and that wall deposition continues despite protoplast retraction and F-actin disruption.

A steroid isolated from the water mold Achlya heterosexualis induces neurogenesis in vitro and in vivo.

Using 22R-hydroxycholesterol as a sub-structure to screen natural compound databases, we identified a naturally occurring steroid (sc-7) with a 16-acetoxy-22R-hydroxycholesterol moiety, in which the hydroxyl groups in positions 3 and 22 are esterified by an acetoxy group and in which the carbon in position 26 carries a functional diacetylamino. sc-7 is an analog of the sex steroids dehydro-oogoniol and antheridiol, can be isolated from the water mold Achlya heterosexualis, and promoted neurogenesis in vitro and in vivo. Mouse embryonic teratocarcinoma P19 cells exposed to sc-7 for 2days followed by a 5-day wash-out differentiated into cholinergic neurons that expressed specific neuronal markers and displayed axonal formation. Axons continued growing up to 28days after treatment. In vivo, infusion of sc-7 for 2weeks into the left ventricle of the rat brain followed by a 3-week wash-out induced bromodeoxyuridine uptake by cells of the ependymal layer and subventricular zone that co-localized with doublecortin and glial fibrillary acidic protein immunostaining, demonstrating induction of proliferation and differentiation of neuronal progenitors. Migrating neuroblasts were also observed in the corpus callosum. Thus, under these experimental conditions, adult ependymal cells resumed proliferation and differentiation. Taken together, these results suggest that sc-7 is an interesting molecule for stimulating in situ neurogenesis from resident neuronal progenitors as part of neuron replacement therapy. sc-7 did not bind to nuclear steroid receptors and was not metabolized as a steroid, supporting our hypothesis that the neurogenic effect of sc-7 is not likely due to a steroid-like effect.

Individual and combined effects of multiple pathogens on Pacific treefrogs.

In nature, individual hosts often encounter multiple pathogens simultaneously, which can lead to additive, antagonistic, or synergistic effects on hosts. Synergistic effects on infection prevalence or severity could greatly affect host populations. However, ecologists and managers often overlook the influence of pathogen combinations on hosts. This is especially true in amphibian conservation, even though multiple pathogens coexist within amphibian populations, and several pathogens have been implicated in amphibian population declines and extinctions. Using an amphibian host, Pseudacris regilla (Pacific treefrog), we experimentally investigated interactive effects among three pathogens: the trematode Ribeiroia sp. (hereafter, Ribeiroia), the fungus Batrachochytrium dendrobatidis (hereafter, BD), and the water mold Achlya flagellata. We detected no effects of A. flagellata, but did find effects of Ribeiroia and BD that varied depending on context. Low doses of Ribeiroia caused relatively few malformations, while higher Ribeiroia doses caused numerous deformities dominated by missing and reduced limbs and limb elements. Exposure to low doses of BD accelerated larval host development, despite there being no detectable BD infections, while exposure to higher BD doses caused infection but did not alter developmental rate. Hosts exposed to both Ribeiroia and BD exhibited the highest mortality, although overall evidence of interactive effects of multiple pathogens was limited. We suggest further research on the influence of multi-pathogen assemblages on amphibians, particularly under a variety of ecological conditions and with a wider diversity of hosts and pathogens.

An investigation of the effects of Ca²+ channel inhibitors on branching and chemotropism in the oomycete Achlya bisexualis: Support for a role for Ca²+ in apical dominance.

In an attempt to better understand branching and chemotropism, we describe the effects of Ca²+ channel inhibitors on these processes in Achlya bisexualis, using a branch induction technique and whole plate assays. Branching appears to be a two step process with the initial formation of a bump from which a branch emerges. Verapamil increased numbers of branches in whole plate assays and decreased the distance from the first branch to the tip. In induction assays verapamil increased the number of bumps formed, although in some hyphae it inhibited the transition from an initial bump to a branch. When a branch formed it did not affect the time taken to branch. It had no effect on chemotropism. Lanthanum (La³+) and gadolinium (Gd³+) also increased branching in whole plate assays but their effect was much less marked and they had no effect on bump/branch number in induction assays. Gd³+ decreased the time taken to branch. Both La³+ and Gd³+ increased chemotropism. These data suggest firstly that the respective inhibitors may affect different parts of the branching process and secondly that Ca²+ influx through channels may not be a requirement for branching, indeed such movements may suppress branching. This would fit with elevated Ca²+ at the tip playing a role in apical dominance.

Genotoxicity of two pathogenic strains of zoosporic fungi (Achlya klebsiana and Aphanomyces laevis) on erythrocytes of Nile tilapia Oreochromis niloticus niloticus.

In the present work we have described the genotoxic potential of two pathogenic strains of zoosporic fungi (Achlya klebsiana and Aphanomyces laevis) on erythrocytes of Nile tilapia Oreochromis niloticus niloticus for the first time by three complementary tests: micronucleus test (MN), nuclear lesions (NL) and comet assay (CA). The groups exposed to the zoosporic fungi subjected to the MN and NL test showed statistically significant differences in MN and NL frequencies with respect to the control one. Also, a significant increase (p<0.001) in micronuclei and nuclear lesions frequencies were recorded with the increase in exposure time. A correlation was observed between the frequencies of MN and NL, suggesting the importance for recording this anomaly in order to improve the information obtained with the MN test. Therefore, our results suggest that the nuclear lesions found here should be considered indicators of genotoxicity, in addition to the typical micronuclei forms. The result of the comet assay showed a significant difference in the percentages of the damaged DNA in the comet tail (%TDNA) of the treated groups comparing to the control. A significant (p<0.001) increase in the DNA damage of the treated groups with the increase in exposure time was recorded, confirming the results of the MN and the NL tests. The results of the current study will be useful for future work involving the biomonitoring of regions where Nile tilapia survive. These data allow us to consider O. niloticus niloticus as a good bioindicator of the effects of genotoxic agents that might be present in the aquatic habitat.

On primordial sense-antisense coding.

The genetic code is implemented by aminoacyl-tRNA synthetases (aaRS). These 20 enzymes are divided into two classes that, despite performing same functions, have nothing common in structure. The mystery of this striking partition of aaRSs might have been concealed in their sterically complementary modes of tRNA recognition that, as we have found recently, protect the tRNAs with complementary anticodons from confusion in translation. This finding implies that, in the beginning, life increased its coding repertoire by the pairs of complementary codons (rather than one-by-one) and used both complementary strands of genes as templates for translation. The class I and class II aaRSs may represent one of the most important examples of such primordial sense-antisense (SAS) coding (Rodin and Ohno, Orig Life Evol Biosph 25:565-589, 1995). In this report, we address the issue of SAS coding in a wider scope. We suggest a variety of advantages that such coding would have had in exploring a wider sequence space before translation became highly specific. In particular, we confirm that in Achlya klebsiana a single gene might have originally coded for an HSP70 chaperonin (class II aaRS homolog) and an NAD-specific GDH-like enzyme (class I aaRS homolog) via its sense and antisense strands. Thus, in contrast to the conclusions in Williams et al. (Mol Biol Evol 26:445-450, 2009), this could indeed be a "Rosetta stone" gene (Carter and Duax, Mol Cell 10:705-708, 2002) (eroded somewhat, though) for the SAS origin of the two aaRS classes.

Achlya spiralis, a new aquatic oomycete with bent oogonial stalks, isolated from the Burgundian region of France.

Achlya spiralis sp. nov. was isolated from water samples collected in the river Tille in the Burgundian region of France. The new oomycete is described, illustrated and compared with related species of the genus Achlya. It is characterized by the presence of smooth-walled oogonia that are usually borne on bent or twisted oogonial stalks; mainly monoclinous, androgynous and diclinous antheridial branches and eccentric oospores which generally do not mature or mature after a long period of time. The internal transcribed spacer (ITS) region of its rRNA is comprised of 671 bases. The taxonomic description of this new species, its comparison with related oomycetes and the sequence of the ITS region of its rRNA are discussed here.

Response of the rainbow trout monocyte/macrophage cell line, RTS11 to the water molds Achlya and Saprolegnia.

The Saprolegniales are responsible for various fish mycoses worldwide and considered the most important fungi afflicting fresh water fish. Saprolegniosis leads to massive epidermal destruction and macrophage recruitment, yet little is known regarding the cytological response of their piscine hosts. The objective of this study was to explore the response of fish macrophage to members of the Saprolegniales using the rainbow trout monocyte/macrophage cell line, RTS11. After 48 h in co-culture, RTS11 demonstrated chemotaxis, adherence and homotypic aggregation to both live and heat-killed fungal spores and mycelia. This aggregation was enhanced when using conditioned media from co-cultured RTS11 and Achlya, suggesting the presence of synergistic effectors of aggregation. Although fungal toxins were not evident, as cells remained viable throughout fungal overgrowth, phagocytosis was inhibited due to large fungal spore size, allowing these molds to evade macrophage defenses. Although class I MH and other viral response genes showed no significant change in expression, calreticulin and interleukin-8 were moderately up-regulated implicating calcium modulation and chemotactic response, respectively. Cyclooxygenase (COX-2) and the cytokines IL-1beta and TNFalpha were strongly up-regulated in the presence of Achlya, while gene expression of the class II major histocompatibility (MH II) receptor and associated molecules appeared down-regulated, suggesting fungal interference of immune function. Previous studies have shown an increased dependence of macrophage in immune function at low temperatures; based upon data presented here, this reduction of macrophage MH II receptor expression and inability to phagocytose spores may limit host response thereby providing increased susceptibility to these opportunistic pathogens.

Pathogenicity studies with the fungi Aphanomyces invadans, Achlya bisexualis, and Phialemonium dimorphosporum: induction of skin ulcers in striped mullet.

Based on isolations from naturally infected fish in Florida, we investigated the role of the fungi Aphanomyces invadans, Achlya bisexualis, and Phialemonium dimorphosporum in the etiology of ulcerative mycosis (UM) in striped mullet Mugil cephalus. We injected healthy striped mullet subcutaneously with secondary zoospores of four oomycete isolates: two concentrations (50 and 115 zoospores/mL) of SJR (an endemic isolate of Aphanomyces invadans in American shad Alosa sapidissima from the St. Johns River); two concentrations each of CAL (25 and 65 zoospores/mL) and ACH (1,400 and 2,000 zoospores/mL; endemic isolates of Aphanomyces invadans and Achlyva bisexualis, respectively, in striped mullet from the Caloosahatchee River); and two concentrations of the ascomycete culture MTZ (2,500 and 3,500 zoospores/mL; endemic isolate of P. dimorphosporum from whirligig mullet M. gyrans in the Matanzas Inlet). All fish injected with either concentration of SJR developed granulomatous ulcers after 8 d and died within 21 d. Eighty percent (8/10) of fish injected with the high dose of CAL developed ulcers after 13 d and died within 28 d, but only 30% (3/10) of fish injected with the low dose of CAL developed ulcers. Four of the ulcerated fish died within 28 d, and the remaining fish were terminated after 32 d. Fish injected with zoospores of Aphanomyces invadans developed ulcers that were grossly and histologically similar to those observed in naturally infected striped mullet with UM from several estuaries or rivers in Florida. These hemorrhagic skin ulcers were characterized by myonecrosis and the presence of mycotic granulomas. None of the fish injected with ACH, MTZ, or sterile water developed ulcers. This study fulfilled Koch's postulates and demonstrated that ulcers could be experimentally induced in striped mullet after exposure via injection to secondary zoospores of an endemic Florida strain of Aphanomyces invadans.

Achlya abortispora, a new oomycete isolated from water samples taken from a water reservoir in Morocco.

Achlya abortispora sp. nov. was found in water and floating organic matter taken form a dam near Rabat, Morocco. The new species is described and compared with other species of the genus. Distinguishing characteristics of A. abortispora are the production of long fusiform sporangia with achlyoid and aplanoid discharge of zoospores; smooth-walled spherical to club-shaped oogonia, which are usually lateral, but at times intercalary, containing 1 to 20 oospheres. The oogonia can also bear 1 to 5 appendages, which may indicate oogonial proliferation. Most of the oospheres do not mature and are thus abortive. The antheridial branches supplying the oogonia are predominantly diclinous, but at times these may be monoclinous and androgynous. Antheridial branches coil and wrap around the oogonia. Morphologic features of the oomycete and the sequence of the ITS region of its rDNA, as well as their comparison with related species, are discussed. This is the first report of the occurrence of a saprolegniaceous oomycete from Morocco.

Invasive hyphal growth: an F-actin depleted zone is associated with invasive hyphae of the oomycetes Achlya bisexualis and Phytophthora cinnamomi.

We have compared F-actin patterns in invasive and non-invasive oomycete hyphae. In Achlya bisexualis an F-actin depleted zone is present in 70% of invasive but only 9% of non-invasive hyphae. In Phytophthora cinnamomi these figures are 74 and 20%, respectively. Thus, the F-actin depleted zone appears to be associated with invasive growth. TEM images indicate that it is unlikely to represent areas of vesicle accumulation. Measurements of turgor indicate no significant increase under invasive conditions (0.65 MPa (invasive) and 0.63 MPa (non-invasive)). Similarly we found no difference in burst pressures (1.04 MPa (invasive) and 1.06 MPa (non-invasive)), although surrounding agarose may lead to overestimates of invasive tip strength. An F-actin depleted zone has the potential, along with wall softening, to increase protrusive force in the absence of turgor increases. Staining of F-actin in hyphae under hyperosmotic conditions suggests that decreases in F-actin at growing tips may also enable non-invasive growth at very low turgor.

A beta4 integrin-like protein co-localises with a phosphotyrosine containing protein in the oomycete Achlya bisexualis: inhibition of tyrosine phosphorylation slows tip growth.

We present immunocytochemical data that indicate the presence of, and a close association between beta4 integrin-like proteins and proteins containing phosphorylated tyrosine residues in the oomycete Achlya bisexualis. When hyphae were plasmolysed, these proteins were present in wall-membrane attachment sites where there was also F-actin. A combination of immunoblots, ELISA, and a coupled enzyme assay suggest that phosphorylation may occur by both autophosphorylation and through the action of a tyrosine kinase. Tyrphostins, which are inhibitors of tyrosine kinases, abolished the anti-phosphotyrosine staining, inhibited the kinase activity, slowed tip growth and affected the organisation of the actin cytoskeleton, in a dose-dependent manner. By analogy with the integrins and associated kinases of the metazoa we suggest that these proteins could contribute to the process of tip growth by providing a means of bidirectional signaling between the cell wall and the cytoplasm.