Arabidopsis EXO70A1 recruits Patellin3 to the cell membrane independent of its role as an exocyst subunit

The exocyst is a well‐known complex which tethers vesicles at the cell membrane before fusion. Whether an individual subunit can execute a unique function is largely unknown. Using yeast‐two‐hybrid (Y2H) analysis, we found that EXO70A1 interacted with the GOLD domain of Patellin3 (PATL3). The direct EXO70A1‐PATL3 interaction was supported by in vitro and in vivo experiments. In Arabidopsis, PATL3‐GFP colocalized with EXO70A1 predominantly at the cell membrane, and PATL3 localization was insensitive to BFA and TryA23. Remarkably, in the exo70a1 mutant, PATL3 proteins accumulated as punctate structures within the cytosol, which did not colocalize with several endomembrane compartment markers, and was insensitive to BFA. Furthermore, PATL3 localization was not changed in the exo70e2, PRsec6 or exo84b mutants. These data suggested that EXO70A1, but not other exocyst subunits, was responsible for PATL3 localization, which is independent of its role in secretory/recycling vesicle‐tethering/fusion. Both EXO70A1 and PATL3 were shown to bind PI4P and PI(4,5)P₂ in vitro. Evidence was obtained that the other four members of the PATL family bound to EXO70A1 as well, and shared a similar localization pattern as PATL3. These findings offered new insights into exocyst subunit‐specific function, and provided data and tools for further characterization of PATL family proteins.     

Characterization of five tomato phospholipase D cDNAs: rapid and specific expression of LePLDβ1 on elicitation with xylanase

Phospholipase D (PLD, EC 3.1.4.4.) has been implicated in a variety of plant processes, including signalling. In Arabidopsis thaliana a PLD gene family has been described and individual members classified into alpha-, beta- and gamma-classes. Here we describe a second PLD gene family in tomato (Lycopersicon esculentum) that includes three alpha- and two beta-classes. Different expression patterns in plant organs were observed for each PLD. In testing a variety of stress treatments on tomato cell suspensions, PLDbeta1 mRNA was found to rapidly and specifically accumulate in response to the fungal elicitor xylanase. The greatest increase was found 2 h after treatment with 100 microg m1(-1) xylanase (ninefold). In vivo PLD activity increased nearly threefold over a 1.5 h period of treatment. When the elicitor was injected into tomato leaves, PLDbeta1 mRNA accumulation peaked at 2 h (threefold increase), before decreasing to background levels within 72 h. Mutant, non-active xylanase was as effective as the active enzyme in eliciting a response, suggesting that xylanase itself, and not the products resulting from its activity, functioned as an elicitor. When chitotetraose was used as elicitor, no PLDbeta1 mRNA accumulation was observed, thus it is not a general response to elicitation. Together these data show that PLD genes are differentially regulated, reflecting potential differences in cellular function. The possibility that PLDbeta1 is a signalling enzyme is discussed.     

The use of karyotyping in the systematics of yeasts

The use of electrophoretic karyotyping in systematics of yeasts is discussed. New data are provided on the karyotypes of the medically important fungiHortaea werneckii, Filobasidiella (=Cryptococcus)neoformans, andMalassezia species.Hortaea werneckii has twelve to eighteen bands of chromosomal DNA, ranging in size between 500 and 2300 kb. The karyotypes ofFilobasidiella neoformans consist of seven to fourteen bands of chromosomal DNA. The varietiesneoformans andbacillispora cannot be separated by their karyotypes, and no obvious correlation was found with serotypes, geography or habitat. All strains ofMalassezia pachydermatis studied have similar karyotypes consisting of five bands, whereas inM. furfur, four different karyotypes are prevalent. However, each of these karyotypes is stable.     

A multi‐colour/multi‐affinity marker set to visualize phosphoinositide dynamics in Arabidopsis

Phosphatidylinositolphosphates (PIPs) are phospholipids that contain a phosphorylated inositol head group. PIPs represent a minor fraction of total phospholipids, but are involved in many regulatory processes, such as cell signalling and intracellular trafficking. Membrane compartments are enriched or depleted in specific PIPs, providing a unique composition for these compartments and contributing to their identity. The precise subcellular localization and dynamics of most PIP species is not fully understood in plants. Here, we designed genetically encoded biosensors with distinct relative affinities and expressed them stably in Arabidopsis thaliana. Analysis of this multi‐affinity ‘PIPline’ marker set revealed previously unrecognized localization of various PIPs in root epidermis. Notably, we found that PI(4,5)P₂ is able to localize PIP₂‐interacting protein domains to the plasma membrane in non‐stressed root epidermal cells. Our analysis further revealed that there is a gradient of PI4P, with the highest concentration at the plasma membrane, intermediate concentration in post‐Golgi/endosomal compartments, and the lowest concentration in the Golgi. Finally, we also found a similar gradient of PI3P from high in late endosomes to low in the tonoplast. Our library extends the range of available PIP biosensors, and will allow rapid progress in our understanding of PIP dynamics in plants.     

Multiple vacuoles in impaired tonoplast trafficking3 mutants are independent organelles

Plant vacuoles are essential and dynamic organelles, and mechanisms of vacuole biogenesis and fusion are not well characterized. We recently demonstrated that Wortmannin, an inhibitor of Phosphatidylinositol 3-Kinase (PI3K), induces the fusion of plant vacuoles both in roots of itt3/vti11 mutant alleles and in guard cells of wild type Arabidopsis and Fava bean. Here we used Fluorescence Recovery After Photobleaching (FRAP) to demonstrate that the vacuoles in itt3/vti11 are independent organelles. Furthermore, we used fluorescent protein reporters that bind specifically to Phosphatidylinositol 3-Phosphate (PtdIns(3)P) or PtdIns(4)P to show that Wortmannin treatments that induce the fusion of vti11 vacuoles result in the loss of PtdIns(3)P from cellular membranes. These results provided supporting evidence for a critical role of PtdIns(3)P in vacuole fusion in roots and guard cells.     

<Emphasis Type="Italic">Cryptococcus randhawai</Emphasis> sp. nov<Emphasis Type="Italic">.,</Emphasis> a novel anamorphic basidiomycetous yeast isolated from tree trunk hollow of <Emphasis Type="Italic">Ficus religiosa</Emphasis> (peepal tree) from New Delhi,India

A novel anamorphic Cryptococcus species is described, which was isolated in New Delhi (India) from decaying wood of a tree trunk hollow of Ficus religiosa. On the basis of sequence analysis of the D1/D2 domains of the 26S rRNA gene and the internally transcribed spacer (ITS)-1 and ITS-2 region sequences, the isolate belonged to the Cryptococcus albidus cluster (Filobasidiales, Tremellomycetes) and was closely related to Cryptococcus saitoi, Cryptococcus cerealis and Cryptococcus friedmannii with 98% sequence identity. Phenotypically, the species differed from C. saitoi with respect to growth temperature (up to 37^oC), presence of a thin capsule, ability to grow in the absence of vitamins, and inability to assimilate citrate and ethylamine. With respect to C. friedmannii, it differed in growth temperature, ability to assimilate lactose, raffinose, l-rhamnose, myo-inositol, and inability to utilize citrate. Furthermore, our isolate also differed from C. cerealis in growth temperature, presence of capsule and inability to assimilate l-sorbose. In view of the above phenotypic differences and unique rDNA sequences, we consider that our isolate represents a new species of Cryptococcus, and therefore, a new species, Cryptococcus randhawai is proposed for this taxon. The type strain J11/2002 has been deposited in the culture collection of the Centraalbureau voor Schimmelcultures (CBS10160) and CABI Biosciences (IMI 393306).