The ARP2/3 complex, acting cooperatively with Class I formins, modulates penetration resistance in Arabidopsis against powdery mildew invasion.

The actin cytoskeleton regulates an array of diverse cellular activities that support the establishment of plant-microbe interactions and plays a critical role in the execution of plant immunity. However, molecular and cellular mechanisms regulating the assembly and rearrangement of actin filaments (AFs) at plant-pathogen interaction sites remain largely elusive. Here, using live-cell imaging, we show that one of the earliest cellular responses in Arabidopsis thaliana upon powdery mildew attack is the formation of patch-like AF structures beneath fungal invasion sites. The AFs constituting actin patches undergo rapid turnover, which is regulated by the actin-related protein (ARP)2/3 complex and its activator, the WAVE/SCAR regulatory complex (W/SRC). The focal accumulation of phosphatidylinositol-4,5-bisphosphate at fungal penetration sites appears to be a crucial upstream modulator of the W/SRC-ARP2/3 pathway-mediated actin patch formation. Knockout of W/SRC-ARP2/3 pathway subunits partially compromised penetration resistance with impaired endocytic recycling of the defense-associated t-SNARE protein PEN1 and its deposition into apoplastic papillae. Simultaneously knocking out ARP3 and knocking down the Class I formin (AtFH1) abolished actin patch formation, severely impaired the deposition of cell wall appositions, and promoted powdery mildew entry into host cells. Our results demonstrate that the ARP2/3 complex and formins, two actin-nucleating systems, act cooperatively and contribute to Arabidopsis penetration resistance to fungal invasion.

Synchrotron FTIR spectromicroscopy as a tool for studying populations and individual living cells of green algae.

Fourier transform infrared (FTIR) spectromicroscopy was used to study individual living cells of three closely-related species of the green algal genus Chlamydomonas. Three types of spectral variation were observed between individual cells within a single culture, as well as between different cultures: variation around a mean, individual outliers, and the presence of subpopulations. By understanding and controlling this variation, we were able to spectroscopically differentiate between the three closely-related species. Spectral differences were confirmed using principal component analysis, leading to an understanding of the biochemical differences between species. This work highlights the additional information obtained by studying individual cells, and has implications for more traditional bulk measurements.

Discovery, isolation, and structure elucidation of dretamycin.

The Candida albicans fitness test is a whole cell screening platform that utilizes a mixed-pool of C. albicans mutants, each of which carries a heterozygous deletion of a particular gene. In the presence of an antifungal inhibitor, a subset of these mutants exhibits a growth phenotype of hypersensitivity or hyposensitivity. Collectively these mutants reflect aspects of the mechanism of action of the compound in question. In the course of screening natural products a culture of Streptomyces sp. MS-1-4 was discovered to produce a compound, dretamycin, which yielded a fitness profile exhibiting significant hypersensitivity of the DRE2 heterozygote and hyposensitivity of the DIP5 heterozygote. Herein we report the production, isolation, and structure elucidation of dretamycin.

Evaluation of biologically mediated changes in oil sands naphthenic acid composition by Chlamydomonas reinhardtii using negative-ion electrospray orbitrap mass spectrometry.

Industrial activity associated with oil-sands extraction in Canada's Athabasca region produces a variety of contaminants of concern, including naphthenic acid fraction components (NAFCs). NAFCs are a complex mixture of organic compounds that are poorly understood both in terms of their chemical composition and effects on the environment. NAFC toxicity in the unicellular green algae Chlamydomonas reinhardtii P.A.Dangeard was correlated with the presence of the algal cell wall. It was suggested that the toxicity of NAFCs in C. reinhardtii was due to surfactant effects. Surfactant-cell wall interactions are specific and governed by the compound class and structure, and by the nature of the biological material. Here, we investigate the effects of wildtype (WT) C. reinhardtii and two cell-wall mutants on specific classes of NAFCs when growing cultures were treated with a 100 mg · L(-1) solution of NAFCs. Changes in the NAFC composition in the media were examined using high resolution mass spectrometry over a period of 4 d. Algal mediated changes in the NAFCs were limited to specific classes of NAFCs. In particular, the removal of large, classical naphthenic acids, with a double bond equivalent of 8, was observed in WT C. reinhardtii cultures. The observed algal mediated changes in NAFC composition would have been masked by low resolution mass spectrometry and highlight the importance of this tool in examining bioremediation of complex mixtures of NAFCs.

Assessment of the effects of oil sands naphthenic acids on the growth and morphology of Chlamydomonas reinhardtii using microscopic and spectromicroscopic techniques.

Naphthenic acid fraction components (NAFCs) are thought to be a primary agent of toxicity in oil sands process waters (OSPWs) produced by industrial activity in Canada's Athabasca oil sands. They are a complex, poorly characterized mixture of compounds whose mechanisms of toxicity are not well understood. In this work, it was discovered that the unicellular green algae Chlamydomonas reinhardtii are much more tolerant of NAFCs than predicted based on comparison to Chlamydomonas spp. isolated from the OSPW tailings ponds, with exponential growth occurring at 100 mg L(-1) NAFC. Two cell wall mutants of C. reinhardtii exhibited greater tolerance to NAFC exposure. NAFC exposure induced changes in growth form and morphology were most pronounced in wild-type cells. Confocal scanning laser microscopy and Fourier-transform infrared spectromicroscopy indicated changes in cell wall surface proteins and their confirmation after exposure to NAFCs. Such alterations of cell wall proteins are consistent with the effects of surfactants on green algae, and indicate a possible role for classic naphthenic acids in the NAFC mixture to cause surfactant-mediated toxicity. The much greater tolerance to NAFCs under laboratory conditions indicates the likelihood that NAFCs do not act alone as agents of toxicity in algae such as C. reinhardtii, rather they seem to act in combination with other environmental factors to potentiate toxicity.

Broadening the spectrum of ß-lactam antibiotics through inhibition of signal peptidase type I.

The resistance of methicillin-resistant Staphylococcus aureus (MRSA) to all ß-lactam classes limits treatment options for serious infections involving this organism. Our goal is to discover new agents that restore the activity of ß-lactams against MRSA, an approach that has led to the discovery of two classes of natural product antibiotics, a cyclic depsipeptide (krisynomycin) and a lipoglycopeptide (actinocarbasin), which potentiate the activity of imipenem against MRSA strain COL. We report here that these imipenem synergists are inhibitors of the bacterial type I signal peptidase SpsB, a serine protease that is required for the secretion of proteins that are exported through the Sec and Tat systems. A synthetic derivative of actinocarbasin, M131, synergized with imipenem both in vitro and in vivo with potent efficacy. The in vitro activity of M131 extends to clinical isolates of MRSA but not to a methicillin-sensitive strain. Synergy is restricted to ß-lactam antibiotics and is not observed with other antibiotic classes. We propose that the SpsB inhibitors synergize with ß-lactams by preventing the signal peptidase-mediated secretion of proteins required for ß-lactam resistance. Combinations of SpsB inhibitors and ß-lactams may expand the utility of these widely prescribed antibiotics to treat MRSA infections, analogous to ß-lactamase inhibitors which restored the utility of this antibiotic class for the treatment of resistant Gram-negative infections.

The PSBP2 protein of Chlamydomonas reinhardtii is required for singlet oxygen-dependent signaling.

In the green alga Chlamydomonas reinhardtii, the cytosolic Glutathione Peroxidase 5 gene (GPX5) is known to be transcriptionally up-regulated in response to singlet oxygen ((1)O(2)). As demonstrated by previous studies, fusion of the promoter region of GPX5 to the Arylsulfatase 2 gene (ARS2) creates an effective reporter system that can be used to monitor (1)O(2)-driven GPX5 expression. This system was also used in this study to generate a stably transformed C. reinhardtii strain which expresses ARS2 in a (1)O(2)-dependent manner, resulting in the synthesis of a functional protein with detectable activity. Using the strain of C. reinhardtii harboring a (1)O(2)-sensitive reporter construct, a secondary mutagenic screen was performed. This allowed identification of mutant cell lines that were unable to up-regulate expression of the GPX5-ARS2 fusion in response to (1)O(2). In one of these lines, the mutation was subsequently localized to the first exon of the PSBP-like gene (PSBP2). The PSBP2 gene is part of a small protein family in C. reinhardtii, also present in all angiosperms studied thus far. While each member of the PSBP protein family contains a similar domain to the PSBP1 protein, which is a member of the oxygen evolving complex of photosystem II (PSII), the PSBP2 protein does not appear to be involved in PSII function, but may function as a sensor and/or signal mediating molecule of the (1)O(2) generated in the chloroplast.

Inactivation of the STT7 gene protects PsaF-deficient Chlamydomonas reinhardtii cells from oxidative stress under high light.

Photosystem I (PSI) utilizes light energy to excite electrons for the reduction of NADP(+) , and like photosystem II, it is sensitive to excess light. When PSI is excited and unable to be reduced by the electron transport chain, the special pair of chlorophyll molecules, P700(+) , will take electrons from neighboring sources leading to cellular damage. A Chlamydomonas reinhardtii mutant, which is defective in the production of the PsaF subunit of PSI, provides an ideal platform for studying the processes involved in protecting PSI from excess light. This strain dies following the exposure to high light (HL) because of photo-oxidative damage. We used a second-site suppressor screen to identify genes involved in protecting PsaF-deficient PSI from excess light. In doing so, we demonstrated that the absence of the STT7 protein, which is required for LHCII phosphorylation and the process of state transitions suppresses the psaF HL-lethal phenotype. On the basis of chlorophyll fluorescence measurements, the psaF mutant has a more reduced plastoquinone pool at a given photosynthetic photon flux density than the wild-type cells. Under these conditions the process of state transitions will become active, resulting in the transfer of phosphorylated LHCII proteins to PSI, further increasing the excitation of PSI. However, in the psaF stt7 double mutant, the LHCII proteins will not be transferred to PSI, and thus the level of PSI excitation will remain lower. This study provides clear genetic evidence that the HL-lethal phenotype of the psaF mutant is because of PSI overexciation.

Mitochondrial and chloroplastic targeting signals of NADP+-dependent isocitrate dehydrogenase.

Many mitochondrial and chloroplast proteins are encoded in the nucleus and subsequently imported into the organelles via active protein transport systems. While usually highly specific, some proteins are dual-targeted to both organelles. In tobacco (Nicotiana tabacum L.), the cDNA encoding the mitochondrial isoform of NADP+-dependent isocitrate dehydrogenase (NADP+-ICDH) contains two translational ATG start sites, suggesting the possibility of tandem targeting signals. In this work, the putative mitochondrial and chloroplastic targeting signals from NADP+-ICDH were fused to a yellow fluorescent protein (YFP) reporter to generate a series of constructs and introduced into tobacco leaves by Agrobacterium-mediated transient transformation. The subsequent sub-cellular locations of the ICDH:YFP fusion proteins were then examined using confocal microscopy. Constructs predicted to be targeted to the chloroplast all localized to the chloroplast. However, this was not the case for all of the constructs that were predicted to be mitochondrial targeted. Although some constructs localized to mitochondria as expected, others appeared to be chloroplast localized. This was attributed to an additional 50 amino acid residues of the mature NADP+-ICDH protein that were present in those constructs, generated from either 'Xanthi' or 'Petit Havana' cultivars of tobacco. The results of this study raise interesting questions regarding the targeting and processing of organellar isoforms of NADP+-ICDH.

Measurement of metabolite formation in single living cells of Chlamydomonas reinhardtii using synchrotron Fourier-Transform Infrared spectromicroscopy.

We demonstrate the capability of synchrotron-based Fourier-Transform Infrared spectromicroscopy to detect metabolite formation in single, living cells of the unicellular algae Chlamydomonas reinhardtii. We show that the high brightness of the source provides a sufficient signal-to-noise ratio to detect small molecular species accumulating in a spot about 15 microm in size. Time resolved measurements are carried out on cells grown heterotrophically under low-light conditions to study the evolution of products of anaerobic metabolism. The formation of small molecular species, including ethanol and at least one carbonyl containing compound, can be detected with a time resolution of the order of one minute.

Temperature-induced greening of Chlorella vulgaris. The role of the cellular energy balance and zeaxanthin-dependent nonphotochemical quenching.

When cells of the green alga Chlorella vulgaris Beij. are transferred from growth at 5 degrees C and an irradiance of 150 micromol photons m(-2) s(-1) to 27 degrees C and the same irradiance, they undergo what is normally considered a high-light to low-light phenotypic change. This involves a 3-fold increase in cellular chlorophyll content with a concomitant increase in light-harvesting complex polypeptide levels. This process appears to occur in response to the cellular capacity to utilize the products of photosynthesis, with the redox state of the plastoquinone pool sensing the cellular energy balance. The phenotypic adjustment can be enhanced or blocked using chemical inhibitors that modulate the redox state of the plastoquinone pool. The functional changes in the photosynthetic apparatus that occurred during the high-light to low-light acclimation were examined with special consideration paid to the paradox that 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated cells, with non-functional photosystem II (PSII), accumulate light-harvesting polypeptides. At the structural and basic functional levels, the light-harvesting complex of the cells treated with DCMU was indistinguishable from that of the untreated, control cells. To examine how PSII was protected in the DCMU-treated cells, we measured the content of xanthophyll-cycle pigments. It appeared that a zeaxanthin-dependent nonphotochemical quenching process was involved in PSII protection during greening in the presence of DCMU. Metabolic inhibitors of mitochondrial respiration were used to examine how the change in cellular energy balance regulates the greening process. Apparently, the mitochondrion acts to supply energy to the chloroplast during greening, and inhibition of mitochondrial respiration diminishes chlorophyll accumulation apparently through an increase in the redox state of the plastoquinone pool.

Cold-regulated cereal chloroplast late embryogenesis abundant-like proteins. Molecular characterization and functional analyses.

Cold acclimation and freezing tolerance are the result of complex interaction between low temperature, light, and photosystem II (PSII) excitation pressure. Previous results have shown that expression of the Wcs19 gene is correlated with PSII excitation pressure measured in vivo as the relative reduction state of PSII. Using cDNA library screening and data mining, we have identified three different groups of proteins, late embryogenesis abundant (LEA) 3-L1, LEA3-L2, and LEA3-L3, sharing identities with WCS19. These groups represent a new class of proteins in cereals related to group 3 LEA proteins. They share important characteristics such as a sorting signal that is predicted to target them to either the chloroplast or mitochondria and a C-terminal sequence that may be involved in oligomerization. The results of subcellular fractionation, immunolocalization by electron microscopy and the analyses of target sequences within the Wcs19 gene are consistent with the localization of WCS19 within the chloroplast stroma of wheat (Triticum aestivum) and rye (Secale cereale). Western analysis showed that the accumulation of chloroplastic LEA3-L2 proteins is correlated with the capacity of different wheat and rye cultivars to develop freezing tolerance. Arabidopsis was transformed with the Wcs19 gene and the transgenic plants showed a significant increase in their freezing tolerance. This increase was only evident in cold-acclimated plants. The putative function of this protein in the enhancement of freezing tolerance is discussed.