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.

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 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.

Two distinct distributions of F-actin are present in the hyphal apex of the oomycete Achlya bisexualis.

We show that two distinct distributions of F-actin are present in the hyphal apex of the oomycete Achlya bisexualis, that have been chemically fixed with a combination of methylglyoxal and formaldehyde and stained with Alexa phalloidin. In approximately one half of the hyphae examined, an F-actin depleted zone within the apical F-actin cap was observed. The remaining hyphae had a continuous apical cap. In live, growing hyphae two types of cytoplasmic organization were observed at the tips, one in which a clear zone was present which may correlate with the F-actin depleted zone, and one where no such clear zone existed which may represent the continuous cap. We suggest that the F-actin depleted zone may be a structural component of the actin network in a subpopulation of oomycete hyphae and may be comparable to similar F-actin depleted zones at the apices of other tip growing cells such as pollen tubes and root hairs. This observation has implications with regard to models of hyphal extension. Hyphae fixed with formaldehyde alone showed continuous apical F-actin caps. Our ability to resolve the F-actin depleted zone likely reflects the cross-linking capabilities of methylglyoxal. The methylglyoxal-formaldehyde combination fixative gave more stained hyphae, brighter staining and more complete staining of F-actin compared to formaldehyde alone.

Molecular cloning and characterization of two different cDNAs encoding the molecular chaperone Hsp90 in the oomycete Achlya ambisexualis.

The chaperone Hsp90 plays a key role in the maturation and activation of many 'client' proteins in eukaryotic cells. In the oomycete Achlya ambisexualis two populations of hsp90 transcripts that differ slightly in size (2.8 and 2.9 kb) are present in heat-shocked mycelia. Only the 2.8 kb transcripts are seen in vegetative mycelia and in mycelia undergoing antheridiol-induced differentiation. Two different hsp90 cDNAs were isolated and characterized. Although nearly identical, an additional eight nucleotide sequence was present at the end of the 3'UTR of one of the two cDNAs. RT-PCR analyses indicated that hsp90 transcripts containing the eight nucleotide extension, were present only in heat-shocked mycelia. Hsp90 transcripts lacking this sequence were present in vegetative mycelia and the levels of these transcripts increased in both heat-shocked and hormone-treated mycelia. Each hsp90 cDNA encoded a nearly identical Hsp90 protein. However, two Hsp90 proteins (86 and 84 kDa) were observed on immunoblots of mycelial proteins. Only one of these, i.e., the 86 kDa protein was detected by an anti-phosphoserine antibody, suggesting that the difference in mass of the two Hsp90 isoforms, was due at least in part, to different levels of phosphoserine residues.