Piriformospora indica, an endophytic fungus of the Sebacinaceae family, colonizes the roots of a wide variety of plant species and promotes their growth, in a manner similar to arbuscular mycorrhizal fungi. The results of the present study demonstrate that the fungus interacts also with the non‐mycorrhizal host Arabidopsis thaliana and promotes its growth. The interaction is detectable by the appearance of a strong autofluorescence in the roots, followed by the colonization of root cells by fungal hyphae and the generation of chlamydospores. Promotion of root growth was detectable even before noticeable root colonization. Membrane‐associated proteins from control roots and roots after cultivation with P. indica were separated by two‐dimensional gel‐electrophoresis and identified by electrospray ionization mass spectrometry and tandem mass spectrometry. Differences were found in the expression of glucosidase II, beta‐glucosidase PYK10, two glutathione‐S‐transferases and several so‐far uncharacterized proteins. Based on conserved domains present in the latter proteins their possible roles in plant–microbe interaction are predicted. Taken together, the present results suggest that the interaction of Arabidopsis thaliana with P. indica is a powerful model system to study beneficial plant–microbe interaction at the molecular level. Furthermore, the successful accommodation of the fungus in the root cells is preceded by protein modifications in the endoplasmatic reticulum as well as at the plasma membrane of the host.
With the recent development of techniques for analyzing transmembrane thylakoid proteins by two-dimensional gel electrophoresis, systematic approaches for proteomic analyses of membrane proteins became feasible. In this study, we established detailed two-dimensional protein maps of Chlamydomonas reinhardtii light-harvesting proteins (Lhca and Lhcb) by extensive tandem mass spectrometric analysis. We predicted eight distinct Lhcb proteins. Although the major Lhcb proteins were highly similar, we identified peptides which were unique for specific lhcbm gene products. Interestingly, lhcbm6 gene products were resolved as multiple spots with different masses and isoelectric points. Gene tagging experiments confirmed the presence of differentially N-terminally processed Lhcbm6 proteins. The mass spectrometric data also revealed differentially N-terminally processed forms of Lhcbm3 and phosphorylation of a threonine residue in the N terminus. The N-terminal processing of Lhcbm3 leads to the removal of the phosphorylation site, indicating a potential novel regulatory mechanism. At least nine different lhca-related gene products were predicted by comparison of the mass spectrometric data against Chlamydomonas expressed sequence tag and genomic databases, demonstrating the extensive variability of the C. reinhardtii Lhca antenna system. Out of these nine, three were identified for the first time at the protein level. This proteomic study demonstrates the complexity of the light-harvesting proteins at the protein level in C. reinhardtii and will be an important basis of future functional studies addressing this diversity.In all eukaryotic oxygenic photosynthetic organisms, lightharvesting chlorophyll a-or b-binding proteins (LHC proteins) function in the collection and transfer of light energy to the reaction centers of photosystem II (PSII) (Lhcb proteins) and photosystem I (PSI) (Lhca proteins). Additionally these proteins are also involved in light dissipation and energy quenching. Therefore, light-harvesting proteins are important components of the photosynthetic machinery that optimize photosynthetic function and minimize photooxidative damage in response to light quantity and quality. It has been known for several years that light-harvesting proteins are products of many genes. This concept is illustrated by a recent analysis of the Arabidopsis genome which revealed that the lhc gene family is composed of more than 20 genes (24). Besides the large number of lhc gene products, posttranslational modifications, such as phophorylation, contribute to even more complexity at the protein level (31, 45). Phosphorylation of the major Lhcb proteins of PSII is important in the process of state transitions. This process leads to a redistribution of excitation energy between PSII and PSI by reorganization of the antennae and thereby regulates energy flow between the photosystems. The importance of phosphorylation for state transitions is shown by the phenotype of the Chlamydomonas reinhardtii Stt7 mutant. This mutant is markedly ...
SummaryThe vesicle-inducing protein in plastids (VIPP1) is essential for the biogenesis of thylakoid membranes in cyanobacteria and plants. VIPP1 and its bacterial ancestor PspA form large homo-oligomeric rings of >1 MDa. We recently demonstrated that VIPP1 interacts with the chloroplast J-domain co-chaperone CDJ2 and its chaperone partner HSP70B, and hypothesized that the chaperones might be involved in the assembly and/or disassembly of VIPP1 oligomers. To test this hypothesis, we analysed the composition of VIPP1/chaperone complexes in Chlamydomonas reinhardtii cell extracts and monitored effects of the chaperones on VIPP1 assembly states in vitro. We found that CGE1, the chloroplast GrpE homologue, is also part of complexes with HSP70B, CDJ2 and VIPP1. We observed that CDJ2-VIPP1 accumulated as low-and high-molecular-weight complexes in ATP-depleted cell extracts, but as intermediate-size complexes in extracts supplemented with ATP. This was consistent with a role for the chaperones in VIPP1 assembly and disassembly. Using purified proteins, we could demonstrate that the chaperones indeed facilitated both the assembly and disassembly of VIPP1 oligomers. Electron microscopy studies revealed that, in contrast to PspA, VIPP1 rings assembled into rod-shaped supercomplexes that were morphologically similar to microtubule-like structures observed earlier in various plastid types. VIPP1 rods, too, were disassembled by the chaperones, and chaperone-mediated rod disassembly also occurred when VIPP1 lacked an approximately 30-aa C-terminal extension present in VIPP1 homologues but absent in PspA. By regulating the assembly state of VIPP1, the chloroplast HSP70 chaperone system may play an important role in the maintenance/biogenesis of thylakoid membranes.
A new high-throughput computational strategy was established that improves genomic data mining from MS experiments. The MS/MS data were analyzed by the SEQUEST search algorithm and a combination of de novo amino acid sequencing in conjunction with an error-tolerant database search tool, operating on a 256 processor computer cluster. The error-tolerant search tool, previously established as GenomicPeptideFinder (GPF), enables detection of intron-split and/or alternatively spliced peptides from MS/MS data when deduced from genomic DNA. Isolated thylakoid membranes from the eukaryotic green alga Chlamydomonas reinhardtii were separated by 1-D SDS gel electrophoresis, protein bands were excised from the gel, digested in-gel with trypsin and analyzed by coupling nano-flow LC with MS/MS. The concerted action of SEQUEST and GPF allowed identification of 2622 distinct peptides. In total 448 peptides were identified by GPF analysis alone, including 98 intron-split peptides, resulting in the identification of novel proteins, improved annotation of gene models, and evidence of alternative splicing.
Cyanobacteria contain several genes, annotated ndh, whose products show sequence similarities to subunits found in complex I (NADH:ubiquinone oxidoreductase) of eubacteria and mitochondria. However, it is still unclear whether the cyanobacterial ndh gene products actually form a single large protein complex or exist as smaller independent complexes. To address this, we have constructed a strain of Synechocystis sp. PCC 6803 in which the C terminus of the NdhJ subunit was fused to an His 6 tag to aid isolation. Three major NdhJ-containing complexes were resolved by blue native polyacrylamide gel electrophoresis, with approximate apparent molecular masses of 460, 330, and 110 kDa. N-terminal sequencing and mass spectrometry revealed that the 460-kDa complex contained ten annotated ndh gene products. Detergent-induced fragmentation experiments indicated that the 460-kDa complex was composed of hydrophobic (150 kDa) and hydrophilic (110 -130 kDa) modules similar to that found in the minimal form of complex I found in Escherichia coli, except that the electron input module was not conserved. The difference in size between the 460-and 330-kDa complexes is attributed to differences in the stoichiometry of the hydrophilic and hydrophobic modules in the complex, either 2:1 or 1:1, respectively. We have also detected the presence of two new Ndh subunits (slr1623 and sll1262) that are unrelated to subunits in the eubacterial complex I but which have homologues in the closely related chloroplast Ndh complex of maize (Funk, E., Schä fer, E., and Steinmü ller, K. (1999) J. Plant Physiol. 154, 16 -23). The presence of these additional subunits might reflect the use by the NDH-1 and Ndh complexes of a different, so far unidentified, electron input module.
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