Browsing by Author "Roitsch, Thomas"

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Cloning and characterization of a novel LpWRKY1 transcription factor in tomato
(Elsevier B.V., 2008) Hofmann, Markus Georg; Sinha, Alok Krishna; Proels, Reinhard Korbinian; Roitsch, Thomas
The initiation of defence responses in plants is accompanied by fundamental changes in gene expression: the expression of pathogenesis- related genes is co-ordinately regulated with metabolic changes such as down regulation of photosynthesis and induction of sink metabolism. To identify candidate regulators of this co-ordinated regulatory mechanism, the role of WRKY transcription factors in the initiation of defence response was analysed in tomato. A WRKY-type transcription factor (LpWRKY1) from tomato was cloned by a reverse Northern approach. The corresponding mRNA is rapidly and transiently induced after challenging the cells with an elicitor-preparation derived from the wilt inducing fungus Fusarium oxysporum lycopersici (E-FOL) and the fungal elicitor chitosan, whereas the endogenous signals systemin and salicylic acid are inactive. Inhibition of protein biosynthesis by cycloheximide results in sustained induction of mRNA for LpWRKY1. In contrast, the transient induction of the gene encoding LpWRKY1 in response to elicitation by E-FOL is inhibited by the protein-kinase inhibitor staurosporine and may be mimicked by the phosphatase inhibitors endothall and cantharidine indicating the involvement of protein phosphorylation in the regulation of WRKY-type transcription factors. Direct proof of this postranslational modification of LpWRKY1 was obtained by demonstrating in-gel kinase assays using recombinant LpWRKY1 as substrate. A 44 kDa and a 67 kDa protein kinase were shown to be transiently activated to phosphorylate LpWRKY1 protein in response to elicitation with E-FOL.
Establishment of a photoautotrophic cell suspension culture of Arabidopsis thaliana for photosynthetic, metabolic, and signaling studies
(Oxford University Press, 2012) Hampp, Christine; Richter, Andreas; Osorio, Sonia; Zellnig, Gunther; Sinha, Alok Krishna; Jammer, Alexandra; Fernie, Alisdair R.; Grimm, Bernhard; Roitsch, Thomas
Plant cell suspension cultures have been used as model sys- tems to circumvent the problems associated with the analyses of a multi-factorial plant that is composed of multiple tissue and cell types exposed to diverse signals. A number of plant suspension cultures have proven to be valuable to study vari- ous topics including defense response, secondary metabolite formation, ion transport, gene regulation, and signal trans- duction (Roitsch and Sinha, 2002 and references therein). How- ever, most cultures reported to date, including the cultures from model species such as Arabidopsis (Christie and Jenkins, 1996), require the presence of sugar in the medium and are characterized by no or negligible photosynthetic activity (Roitsch and Sinha, 2002). For only a very limited number of species, such as Chenopodium rubrum, have true photoauto- trophic (PA) cultures been established (see references in Roitsch and Sinha, 2002). Such cultures combine the advan- tages of plant suspension cultures with carbon autotrophy and represent very powerful model systems for plant research. Unfortunately, for as-yet unknown reasons, it is very difficult to establish PA cultures (Widholm, 1992); hence, PA cultures from only a few crop species have been established. PA cul- tures have, however, been used to address various aspects of photosynthesis, herbicide effects, and secondary product formation from exclusively chloroplast localized pathways as well as in characterization of the metabolic changes occurring during the source–sink transition (Roitsch et al., 1995) and the coordinated regulation of primary metabolism and defense responses (Ehness et al., 1997). In parallel, a large number of mutant and transgenic Arabidopsis plants have been char- acterized with respect to their physiology, biochemistry, and molecular biology. That said, the establishment of a PA cell culture of Arabidopsis has, to date, proven elusive.
Plant physiology meets phytopathology: relations between plant primary metabolism and plant-pathogen-interactions
(Oxford University Press, 2007) Berger, Susanne; Sinha, Alok Krishna; Roitsch, Thomas
Phytopathogen infection leads to changes in secondary metabolism based on the induction of defence programmes as well as to changes in primary metabolism which affect growth and development of the plant. Therefore, pathogen attack causes crop yield losses even in interactions which do not end up with disease or death of the plant. While the regulation of defence responses has been intensively studied for decades, less is known about the effects of pathogen infection on primary metabolism. Recently, interest in this research area has been growing, and aspects of photosynthesis, assimilate partitioning, and source– sink regulation in different types of plant–pathogen interactions have been investigated. Similarly, phytopathological studies take into consideration the physiological status of the infected tissues to elucidate the fine-tuned infection mechanisms. The aim of this review is to give a summary of recent advances in the mutual interrelation between primary metabolism and pathogen infection, as well as to indicate current developments in non-invasive techniques and important strategies of combining modern molecular and physiological techniques with phytopathology for future investigations.
Post-translational depression of invertase activity in source leaves via down regulation of invertase inhibitor expression is part of the plant defense response
(Oxford University Press, 2010) Bonfig, Katharina B.; Gabler, Andrea; Simon, Uwe K.; Luschin-Ebengreuth, Nora; Hatz, Martina; Berger, Susanne; Muhammad, Naseem; Zeier, Jurgen; Sinha, Alok Krishna; Roitsch, Thomas
There is increasing evidence that pathogens do not only elicit direct defense responses, but also cause pronounced changes in primary carbohydrate metabolism. Cell-wall-bound invertases belong to the key regulators of carbohydrate partitioning and source-sink relations. Whereas studies have focused so far only on the transcriptional induction of invertase genes in response to pathogen infection, the role of post-translational regulation of invertase activity has been neglected and was the focus of the present study. Expression analyses revealed that the high mRNA level of one out of three proteinaceous invertase inhibitors in source leaves of Arabidopsis thaliana is strongly repressed upon infection by a virulent strain of Pseudomonas syringae pv. tomato DC3000. This repression is paralleled by a decrease in invertase inhibitor activity. The physiological role of this regulatory mechanism is revealed by the finding that in situ invertase activity was detectable only upon infection by P. syringae. In contrast, a high invertase activity could be measured in vitro in crude and cell wall extracts prepared from both infected and non-infected leaves. The discrepancy between the in situ and in vitro invertase activity of control leaves and the high in situ invertase activity in infected leaves can be explained by the pathogen-dependent repression of invertase inhibitor expression and a concomitant reduction in invertase inhibitor activity. The functional importance of the release of invertase from post-translational inhibition for the defense response was substantiated by the application of the competitive chemical invertase inhibitor acarbose. Post-translational inhibition of extracellular invertase activity by infiltration of acarbose in leaves was shown to increase the susceptibility to P. syringae. The impact of invertase inhibition on spatial and temporal dynamics of the repression of photosynthesis and promotion of bacterial growth during pathogen infection supports a role for extracellular invertase in plant defense. The acarbose-mediated increase in susceptibility was also detectable in sid2 and cpr6 mutants and resulted in slightly elevated levels of salicylic acid, demonstrating that the effect is independent of the salicylic acid-regulated defense pathway. These findings provide an explanation for high extractable invertase activity found in source leaves that is kept inhibited in situ by post-translational interaction between invertase and the invertase inhibitor proteins. Upon pathogen infection, the invertase activity is released by repression of invertase inhibitor expression, thus linking the local induction of sink strength to the plant defense response.
Role of α-tocopherol in cellular signaling: α-tocopherol inhibits stress-induced mitogen activated protein kinase activation
(Springer Science, 2011) Hyun, Tae Kyung; Kumar, Kundan; Rao, Kudupudi Prabhakara; Sinha, Alok Krishna; Roitsch, Thomas
Tocopherols belong to the plant-derived poly phenolic compounds known for antioxidant functions in plants and animals. Activation of mitogen-activated protein kinases (MAPK) is a common reaction of plant cells in defense-related signal transduction pathways. We report a novel non-antioxidant function of α-tocopherol in higher plants linking the physiological role of tocopherol with stress signalling pathways. Pre-incubation of a low concentration of 50 μM α-tocopherol negatively interferes with MAPK activation in elicitor-treated tobacco BY2 suspension culture cells and wounded tobacco leaves, whereas pre-incubated BY2 cells with α-tocopherol phosphate did not show the inhibitory effect on stimuli-induced MAPK activation. The decreased MAPK activity was neither due to a direct inhibitory effect of α-tocopherol nor due to the induction of an inhibitory or inactivating activity directly affecting MAPK activity. The data support that the target of α-tocopherol negatively regulates an upstream component of the signaling pathways that leads to stress dependent MAPK activation.
Tomato mitogen activated protein kinases regulate the expression of extracellular invertase Lin6 in response to stress related stimuli
(CSIRO Publishing, 2009) Hyun, Tae Kyung; Hoffmann, Anja; Sinha, Alok Krishna; Roitsch, Thomas
Activation of mitogen-activated protein kinases (MAPKs) is a common reaction of plant cells in defence-related signal transduction pathways. Since the downstream events after the activation of MAPKs are largely unknown in plants, the role of MAPKs in the coordinate regulation of defence reactions and primary carbon metabolism by stress related stimuli has been analysed in tomato (Lycopersicon peruvianum Mill.). Thus, the relationship between MAPK, LpMPK2 and LpMPK3 and extracellular invertase Lin6, as the key enzyme of an apoplasmic phloem unloading pathway, has been analysed. It was observed that the mRNAs of LpMPK3 and Lin6 are sequentially induced by the same set of stress related stimuli, wounding, a fungal elicitor derived from Fusarium oxysporum lycopersici, the endogenous plant derived elicitor PGA and salt stress, while LpMPK2 transcripts are constitutively expressed. In a gain of function approach, a His-tagged version of LpMPK2 and a HA-tagged version of LpMPK3 were transiently and functionally expressed in leaves of transgenic tobacco (Nicotiana tabacum L.) plants expressing the β-glucuronidase reporter gene under control of the Lin6 promoter via agro-infection. The induction of the Lin6 promoter, as revealed by an increase in β-glucuronidase activity after 24 h, was dependent both on the expression and activation of both LpMPK2 and LpMPK3. These data suggest that the induction of extracellular invertase Lin6 by stress-related stimuli requires LpMPK2 and LpMPK3, and thus demonstrate that MAPK signalling might be involved in the regulation of primary carbon metabolism in general and sink metabolism in particular.