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.

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.

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.