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Cellular Responses of the Late Blight Pathogen Phytophthora infestans to Cyclic Lipopeptide Surfactants and Their Dependence on G Proteins
Mortel, J.E. van de; Tran, H. ; Govers, F. ; Raaijmakers, J.M. - \ 2009
Applied and Environmental Microbiology 75 (2009)15. - ISSN 0099-2240 - p. 4950 - 4957.
pseudomonas-fluorescens dr54 - channel-forming activities - alpha-subunit - bacillus-subtilis - model membranes - pythium-ultimum - beta-subunit - syringomycin - fungi - biosynthesis
Oomycete pathogens cause major yield losses for many crop plants, and their control depends heavily on agrochemicals. Cyclic lipopeptides (CLPs) were recently discovered as a new class of natural compounds with strong activities against oomycetes. The CLP massetolide A (Mass A), produced by Pseudomonas fluorescens, has zoosporicidal activity, induces systemic resistance, and reduces late blight in tomato. To gain further insight into the modes of action of CLPs, the effects of Mass A on pore formation, mycelial growth, sporangium formation, and zoospore behavior were investigated, as was the involvement of G proteins in the sensitivity of Phytophthora infestans to Mass A. The results showed that Mass A induced the formation of transmembrane pores with an estimated size of between 1.2 and 1.8 nm. Dose-response experiments revealed that zoospores were the most sensitive to Mass A, followed by mycelium and cysts. Mass A significantly reduced sporangium formation and caused increased branching and swelling of hyphae. At relatively low concentrations, Mass A induced encystment of zoospores. It had no effect on the chemotactic response of zoospores but did adversely affect zoospore autoaggregation. A loss-of-function transformant of P. infestans lacking the G-protein subunit was more sensitive to Mass A, whereas a gain-of-function transformant required a higher Mass A concentration to interfere with zoospore aggregation. Results indicate that Mass A disturbs various developmental stages in the life cycle of P. infestans and suggest that the cellular responses of P. infestans to this CLP are, in part, dependent on G-protein signaling
A Phytophthora sojae G protein alpha subunit is involved in chemotaxis to soybean isoflavones
Hua, C. ; Wang, Y. ; Zheng, X. ; Dou, D. ; Zhang, Z. ; Govers, F. - \ 2008
Eukaryotic Cell 7 (2008)12. - ISSN 1535-9778 - p. 2133 - 2140.
transcription factor - zoospore encystment - hyphal growth - saccharomyces-cerevisiae - signal-transduction - cyst germination - botrytis-cinerea - beta-subunit - infestans - oomycete
For the soybean pathogen Phytophthora sojae, chemotaxis of zoospores to isoflavones is believed to be critical for recognition of the host and for initiating infection. However, the molecular mechanisms underlying this chemotaxis are largely unknown. To investigate the role of G-protein and calcium signaling in chemotaxis, we analyzed the expression of several genes known to be involved in these pathways and selected one that was specifically expressed in sporangia and zoospores but not in mycelium. This gene, named PsGPA1, is a single-copy gene in P. sojae and encodes a G-protein subunit that shares 96% identity in amino acid sequence with that of Phytophthora infestans. To elucidate the function, expression of PsGPA1 was silenced by introducing antisense constructs into P. sojae. PsGPA1 silencing did not disturb hyphal growth or sporulation but severely affected zoospore behavior, including chemotaxis to the soybean isoflavone daidzein. Zoospore encystment and cyst germination were also altered, resulting in the inability of the PsGPA1-silenced mutants to infect soybean. In addition, the expressions of a calmodulin gene, PsCAM1, and two calcium- and calmodulin-dependent protein kinase genes, PsCMK3 and PsCMK4, were increased in the mutant zoospores, suggesting that PsGPA1 negatively regulates the calcium signaling pathways that are likely involved in zoospore chemotaxis
Isolation and characterization of a tomato non-specific lipid transfer protein involved in polygalacturonase-mediated pectin degradation
Tomassen, M.M.M. ; Barrett, D.M. ; Valk, H.C.P.M. van der; Woltering, E.J. - \ 2007
Journal of Experimental Botany 58 (2007)5. - ISSN 0022-0957 - p. 1151 - 1160.
beta-subunit - gene-expression - transgenic tomatoes - fruit - isoenzymes - plant - converter - ultrastructure - manipulation - conversion
An important aspect of the ripening process of tomato fruit is softening. Softening is accompanied by hydrolysis of the pectin in the cell wall by pectinases, causing loss of cell adhesion in the middle lamella. One of the most significant pectin-degrading enzymes is polygalacturonase (PG). Previous reports have shown that PG in tomato may exist in different forms (PG1, PG2a, PG2b, and PGx) commonly referred to as PG isoenzymes. The gene product PG2 is differentially glycosylated and is thought to associate with other proteins to form PG1 and PGx. This association is thought to modulate its pectin-degrading activity in planta. An 8 kDa protein that is part of the tomato PG1 multiprotein complex has been isolated, purified, and functionally characterized. This protein, designated `activator¿ (ACT), belongs to the class of non-specific lipid transfer proteins (nsLTPs). ACT is capable of `converting¿ the gene product PG2 into a more active and heat-stable form, which increases PG-mediated pectin degradation in vitro and stimulates PG-mediated tissue breakdown in planta. This finding suggests a new, not previously identified, function for nsLTPs in the modification of hydrolytic enzyme activity. It is proposed that ACT plays a role in the modulation of PG activity during tomato fruit softening.
The chicken pituitary-specific transcription factor Pit-1 is involved in the hypothalamic regulation of pituitary hormones
As, P. van; Janssens, K. ; Pals, K. ; Groef, B. De; Onagbesan, O.M. ; Bruggeman, V. ; Darras, V.M. ; Denef, C. ; Decuypere, E. - \ 2006
Acta Veterinaria Hungarica 54 (2006)4. - ISSN 0236-6290 - p. 455 - 471.
thyrotropin-releasing-hormone - thyroid-stimulating hormone - growth-hormone - molecular-cloning - gene-expression - beta-subunit - sequence-analysis - receptor - secretion - protein
Pit-1 is a pituitary-specific POU-domain DNA binding factor, which binds to and trans-activates promoters of growth hormone- (GH), prolactin- (PRL) and thyroid stimulating hormone-beta- (TSHbeta) encoding genes. Thyrotropin-releasing hormone (TRH) is located in the hypothalamus and stimulates TSH, GH and PRL release from the pituitary gland. In the present study, we successfully used the cell aggregate culture system for chicken pituitary cells to study the effect of TRH administration on the ggPit-l* (chicken Pit-1), GH and TSHbeta mRNA expression in vitro. In pituitary cell aggregates of 11-day-old male broiler chicks the ggPit-l * mRNA expression was significantly increased following TRH administration, indicating that the stimulatory effects of TRH on several pituitary hormones are mediated via its effect on the ggPit-l* gene expression. Therefore, a semiquantitative RT-PCR method was used to detect possible changes in GH and TSHbeta mRNA levels. TRH affected both the GH and TSHbeta mRNA levels. The results of this in vitro study reveal that ggPit-1 * has a role in mediating the stimulatory effects of TRH on pituitary hormones like GH and TSHbeta in the chicken pituitary.
Downstream targets of the Phytophthora infestans G alpha subunit PiGPA1 revealed by cDNA-AFLP
Dong, W. ; Latijnhouwers, M. ; Jiang, R.H.Y. ; Meijer, H.J.G. ; Govers, F. - \ 2004
Molecular Plant Pathology 5 (2004)5. - ISSN 1464-6722 - p. 483 - 494.
gene-expression profiles - gtp-binding proteins - beta-subunit - sequences - mutations - infection - database
In many plant pathogens heterotrimeric G-proteins are essential signalling components involved in development and pathogenicity. In the late blight oomycete pathogen Phytophthora infestans the G-protein (x subunit PiGPA1 controls zoospore motility and is required for virulence. To identify G-protein targets and signalling pathways downstream of PiGPA1, we used an optimized cDNA-AFLP protocol for analysing gene expression profiles in hypovirulent P. infestans strains that were previously generated by silencing the Pigpa1 gene. First, expression profiles in sporangia and mycelium of the wild-type strain were compared, and this revealed a substantial number of mycelium- or sporangia-specific transcript derived fragments (TDFs). Subsequently, profiles in sporangia of wild-type, Pigpa1-silenced mutants and of a strain expressing a constitutively active form of PiGPA1 were compared. From a total of 2860 TDFs, 92 were down- and 19 upregulated in the Pigpa1-silenced mutants. A subset of the differential TDFs was cloned and sequenced, and homology searches were carried out against Phytophthora EST and genomic databases and the NCBI database. cDNA-AFLP expression profiles were verified by Northern blot analysis or RT-PCR. The power of cDNA-AFLP for the identification of target genes in knock-down or gain-of-function mutants is discussed.
A G alpha subunit controls zoospore motility and virulence in the potato late blight pathogen Phytophthora infestans
Latijnhouwers, M. ; Ligterink, J.W. ; Vleeshouwers, V.G.A.A. ; West, P. van; Govers, F. - \ 2004
Molecular Microbiology 51 (2004)4. - ISSN 0950-382X - p. 925 - 936.
heterotrimeric g-proteins - beta-subunit - arabidopsis-thaliana - fungus phytophthora - cryphonectria-parasitica - signal-transduction - cell-proliferation - neurospora-crassa - gamma-subunit - genes
The heterotrimeric G-protein pathway is a ubiquitous eukaryotic signalling module that is known to regulate growth and differentiation in many plant pathogens. We previously identified Pigpa1, a gene encoding a G-protein alpha subunit from the potato late blight pathogen Phytophthora infestans. P. infestans belongs to the class oomycetes, a group of organisms in which signal transduction processes have not yet been studied at the molecular level. To elucidate the function of Pigpa1, PiGPA1-deficient mutants were obtained by homology-dependent gene silencing. The Pigpa1-silenced mutants produced zoospores that turned six to eight times more frequently, causing them to swim only short distances compared with wild type. Attraction to the surface, a phenomenon known as negative geotaxis, was impaired in the mutant zoospores, as well as autoaggregation and chemotaxis towards glutamic and aspartic acid. Zoospore production was reduced by 20-45% in different Pigpa1-silenced mutants. Transformants expressing constitutively active forms of PiGPA1, containing amino acid substitutions (R177H and Q203L), showed no obvious phenotypic differences from the wild-type strain. Infection efficiencies on potato leaves ranged from 3% to 14% in the Pigpa1-silenced mutants, compared with 77% in wild type, showing that virulence is severely impaired. The results prove that PiGPA1 is crucial for zoospore motility and for pathogenicity in an important oomycete plant pathogen.