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Stephen C. Winans - One of the best experts on this subject based on the ideXlab platform.
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Agrobacterium tumefaciens can obtain sulphur from an opine that is synthesized by octopine synthase using S-methylmethionine as a substrate
Molecular Microbiology, 2012Co-Authors: Ana L. Flores-mireles, Anatol Eberhard, Stephen C. WinansAbstract:Agrobacterium tumefaciens incites Plant Tumors that produce nutrients called opines, which are utilized by the bacteria during host colonization. Various opines provide sources of carbon, nitrogen, and phosphorous, but virtually nothing was previously known about how A. tumefaciens acquires sulfur during colonization. Some strains encode an operon required for the catabolism of the opine octopine. This operon contains a gene, msh, that is predicted to direct the conversion of S-methylmethionine (SMM) and homocysteine (HCys) to two equivalents of methionine. Purified Msh carried out this reaction, suggesting that SMM could be an intermediate in opine catabolism. Purified octopine synthase (Ocs, normally expressed in Plant Tumors) utilized SMM and pyruvate to produce a novel opine, designated sulfonopine, whose catabolism by the bacteria would regenerate SMM. Sulfonopine was produced by tobacco and Arabidopsis when colonized by A. tumefaciens and was utilized as sole source of sulfur by A. tumefaciens. Purified Ocs also used thirteen other proteogenic and nonproteogenic amino acids as substrates, including three that contain sulfur. Sulfonopine and eleven other opines were tested for induction of octopine catabolic operon and all were able to do so. This is the first study of the acquisition of sulfur, an essential element, by this pathogen.
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Constitutive Mutations of the OccR Regulatory Protein Affect DNA Bending in Response to Metabolites Released from Plant Tumors
The Journal of biological chemistry, 2001Co-Authors: Reiko Akakura, Stephen C. WinansAbstract:OccR is a LysR-type transcriptional regulator of Agrobacterium tumefaciens that positively regulates the octopine catabolism operon of the Ti plasmid and is also an autorepressor. Positive control of the occ genes occurs in response to octopine, a nutrient released from crown gall Tumors. OccR binds to a site upstream of the occQ promoter in the presence and absence of octopine. Octopine causes prebound OccR to undergo a conformational change at the DNA binding site that causes changes in footprint length and DNA bending. To determine the roles of these conformational changes in transcriptional activation, we isolated 22 OccR mutants that were able to activate the occQ promoter in the absence of octopine. Thirteen of these mutants contained single amino acid substitutions, and nine contained two base pair changes resulting in two amino acid substitutions, which in most cases acted synergistically. These mutations spanned the entire length of the protein. Most of these mutant proteins in the absence of octopine displayed DNA binding and bending properties characteristic of transcriptionally active OccR-octopine complexes.
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the bases of crown gall tumorigenesis
Journal of Bacteriology, 2000Co-Authors: Jun Zhu, Paul J. J. Hooykaas, Stephen K. Farrand, Philippe Oger, Barbara Schrammeijer, Stephen C. WinansAbstract:The nine decades since Smith and Townsend demonstrated that Agrobacterium tumefaciens causes Plant Tumors (95) have been marked by a series of surprises. Among the most important of these was the report in 1958 that these Tumors could be excised and propagated in vitro without exogenous Plant hormones (7). Equally important were a series of reports beginning about the same time that Tumors released compounds that agrobacteria could use as nutrients (24). Perhaps the most exciting discoveries, reported in the 1970s and 1980s, were that tumorigenesis required the transfer of fragments of oncogenic DNA to infected Plant cells (10), that this process evolved from a conjugal transfer system (99), and that the genes that direct this process are expressed in response to host-released chemical signals (47). This DNA transfer process has become a cornerstone of Plant molecular genetics. The genus Agrobacterium also has provided excellent models for several aspects of host-pathogen interactions, including intercellular transport of macromolecules (11), bacterial detection of host organisms (47), targeting of proteins to Plant cell nuclei (3), and interbacterial chemical signaling via autoinducer-type pheromones (120).
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diverse roles of agrobacterium ti plasmid borne genes in the formation and colonization of Plant Tumors
1999Co-Authors: Stephen C. Winans, Virginia S Kalogeraki, Santina Jafri, Reiko AkakuraAbstract:This chapter describes the various steps in Plant colonization, including binding of the bacteria to host cells, recognition of diffusible host-released chemical signals, and processing and transfer of oncogenic DNA (T-DNA). It includes a discussion on the utilization of tumor-released compounds called opines and on the horizontal transfer of the Ti plasmid. Species of Agrobacterium have been classified primarily by their phytopathogenic properties, which are largely due to differences in plasmid content. With respect to chromosomally encoded properties, A. rubi and A. vitis are well-defined separate taxa whereas A. tumefaciens, A. radiobacter, and A. rhizogenes are not. The most thoroughly studied A. tumefaciens strains contain either octopine-type or nopaline-type Ti plasmids. Virtually all genes found on Ti plasmids play direct or indirect roles in some aspect of crown gall tumorigenesis or tumor colonization. VirD4 is absolutely required for transfer and pilus formation. Some members of the vir regulon are not essential for tumorigenesis and may play other roles in pathogenesis. Since Ti plasmids encode both TraI and TraR, each conjugal donor takes a census of other donors rather than of recipients. It is a challenge even to speculate about the adaptive value of such a system. The observation that two opines regulate conjugation in opposite ways is equally perplexing.
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a luxr luxi type regulatory system activates agrobacterium ti plasmid conjugal transfer in the presence of a Plant tumor metabolite
Journal of Bacteriology, 1994Co-Authors: W C Fuqua, Stephen C. WinansAbstract:Conjugal transfer of Agrobacterium octopine-type Ti plasmids is activated by octopine, a metabolite released from Plant Tumors. Octopine causes conjugal donors to secrete a pheromone, Agrobacterium autoinducer (AAI), and exogenous AAI further stimulates conjugation. The putative AAI synthase and an AAI-responsive transcriptional regulator were found to be encoded by the Ti plasmid traI and traR genes, respectively, and the expression of traR was induced by octopine. The octopine-type traR gene product is highly homologous to the TraR protein recently characterized from a nopaline-type Ti plasmid. TraR and TraI are homologous to the LuxR and LuxI regulatory proteins of Vibrio fischeri, and AAI is similar in structure to the diffusable V. fischeri autoinducer, the inducing ligand of LuxR. TraR activated target genes in the presence of AAI and also activated traR and traI themselves, creating two positive-feedback loops. TraR-AAI-mediated activation in wild-type Agrobacterium strains was dramatically enhanced by culturing on solid media, suggesting a possible role in cell density sensing.
Denis Faure - One of the best experts on this subject based on the ideXlab platform.
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The biotroph Agrobacterium tumefaciens thrives in Tumors by exploiting a wide spectrum of Plant host metabolites
New Phytologist, 2019Co-Authors: Almudena Gonzalez-mula, Joy Lachat, Léo Mathias, Delphine Naquin, Florian Lamouche, Peter Mergaert, Denis FaureAbstract:Agrobacterium tumefaciens is a niche-constructing biotroph that exploits host Plant metabolites. We combined metabolomics, transposon-sequencing (Tn-seq), transcriptomics and reverse genetics to characterize A. tumefaciens pathways involved in the exploitation of resources from the Solanum lycopersicum host Plant. Metabolomics of healthy stems and Plant Tumors revealed the common (e.g., sucrose, glutamate) and enriched (e.g., opines, GABA, GHB, pyruvate) metabolites that A. tumefaciens could use as nutrients. Transposon-sequencing and transcriptomics pinpointed the genes that are crucial and/or up-regulated when the pathogen grew on either sucrose (pgi, kdgA, pycA, cisY) or GHB (blcAB, pckA, eno, gpsA) as a carbon source. While sucrose assimilation involved the Entner-Doudoroff and tricarboxylic acid (TCA) pathways, GHB degradation required the blc genes, tricarboxylic acid cycle and gluconeogenesis. The tumor-enriched metabolite pyruvate is at the node connecting these pathways. Using reverse genetics, we showed that the blc, pckA and pycA loci were important for aggressiveness (tumor weight), proliferation (bacterial charge) and/or fitness (competition between the constructed mutants and wild-type) of A. tumefaciens in Plant Tumors. This work highlighted how a biotroph mobilizes its central metabolism for exploiting a wide diversity of resources in Plant host. It further shows the complementarity of functional genome-wide scans by transcriptomics and transposon-sequencing to decipher the lifestyle of a Plant pathogen. This article is protected by copyright. All rights reserved.
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Integrative and deconvolution omics approaches to uncover the Agrobacterium tumefaciens lifestyle in Plant Tumors
Plant Signaling and Behavior, 2019Co-Authors: Almudena Gonzalez-mula, Marta Torres, Denis FaureAbstract:Agrobacterium tumefaciens is a Plant pathogen which provokes galls on roots and stems (crown-gall disease) and colonizes them. Two approaches combining omics were used to decipher the lifestyle of A. tumefaciens in Plant Tumors: an integrative approach when omics were used on A. tumefaciens cells collected from Plant Tumors, a deconvolution approach when omics were used on A. tumefaciens cells exploiting a single tumor metabolite in pure culture assay. This addendum highlights some recent results on the biotroph lifestyle of A. tumefaciens in Plant Tumors.
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structural basis for high specificity of amadori compound and mannopine opine binding in bacterial pathogens
Journal of Biological Chemistry, 2016Co-Authors: Loic Marty, Magali Aumontnicaise, Yves Dessaux, Denis Faure, Armelle Vigouroux, Serni MoreraAbstract:Agrobacterium tumefaciens pathogens genetically modify their host Plants to drive the synthesis of opines in Plant Tumors. Opines are either sugar phosphodiesters or the products of condensed amino acids with ketoacids or sugars. They are Agrobacterium nutrients and imported into the bacterial cell via periplasmic-binding proteins (PBPs) and ABC-transporters. Mannopine, an opine from the mannityl-opine family, is synthesized from an intermediate named deoxy-fructosyl-glutamine (DFG), which is also an opine and abundant Amadori compound (a name used for any derivative of aminodeoxysugars) present in decaying Plant materials. The PBP MotA is responsible for mannopine import in mannopine-assimilating agrobacteria. In the nopaline-opine type agrobacteria strain, SocA protein was proposed as a putative mannopine binding PBP, and AttC protein was annotated as a mannopine binding-like PBP. Structural data on mannityl-opine-PBP complexes is currently lacking. By combining affinity data with analysis of seven x-ray structures at high resolution, we investigated the molecular basis of MotA, SocA, and AttC interactions with mannopine and its DFG precursor. Our work demonstrates that AttC is not a mannopine-binding protein and reveals a specific binding pocket for DFG in SocA with an affinity in nanomolar range. Hence, mannopine would not be imported into nopaline-type agrobacteria strains. In contrast, MotA binds both mannopine and DFG. We thus defined one mannopine and two DFG binding signatures. Unlike mannopine-PBPs, selective DFG-PBPs are present in a wide diversity of bacteria, including Actinobacteria, α-,β-, and γ-proteobacteria, revealing a common role of this Amadori compound in pathogenic, symbiotic, and opportunistic bacteria.
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The Plant GABA signaling downregulates horizontal transfer of the Agrobacterium tumefaciens virulence plasmid.
New Phytologist, 2016Co-Authors: Julien Lang, Almudena Gonzalez-mula, Ludivine Taconnat, Gilles Clement, Denis FaureAbstract:In the tumor-inducing (Ti) Agrobacterium tumefaciens, quorum sensing activates the horizontal transfer of the virulent Ti plasmid. In pure culture, this process can be impaired by the A. tumefaciens BlcC lactonase, whose expression is induced by gamma-aminobutyrate (GABA). It was therefore hypothesized that host GABA content might modulate quorum sensing and virulence gene dissemination during A. tumefaciens infection. We examined GABA metabolism and transport in Arabidopsis thaliana Tumors combining transcriptomic, metabolomic and histological approaches. In addition, using genetically modified Plants and bacteria, we evaluated the impact of Plant host GABA content on Ti plasmid dissemination. The results showed that GABA and free proline, which acts as an antagonist of GABA uptake in A. tumefaciens, accumulated in wild-type Tumors relative to uninfected Plant tissues. Moreover, comparisons of Tumors induced on Col-0 and her1 Plants showed that the increase in the Plant GABA : proline ratio was associated with both the upregulated expression of the blcC gene and the decreased dissemination of Ti plasmid in tumor-colonizing A. tumefaciens populations. This work demonstrates experimentally that the variation in the GABA content in Plant Tumors can interfere with the dissemination of A. tumefaciens Ti plasmids, and therefore highlights Plant GABA content as an important trait in the struggle against pathogenic bacteria.
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different regulation and roles of lactonases aiib and attm in agrobacterium tumefaciens c58
Molecular Plant-microbe Interactions, 2009Co-Authors: Elise Haudecoeur, Yves Dessaux, Melanie Tannieres, Amelie Cirou, Aurelie Raffoux, Denis FaureAbstract:The phytopathogen Agrobacterium tumefaciens C58 expresses two lactonases, AttM and AiiB. We showed that expression of the aiiB gene was controlled by agrocinopines A and B and required the agrocinopine-ABC transporter Acc, but was not affected by the level of quorum-sensing (QS) signal 3-oxo-octanoylhomoserine lactone (OC8-HSL). In the presence of agrocinopines, a constructed aiiB mutant accumulated OC8-HSL at a level 10-fold higher than that of the wild-type strain, and showed an exacerbated expression of a key QS-regulated function, conjugation of Ti plasmid (in vitro and in Planta), as well as an increase of the number of emerging Tumors on the host Plant. The expression and acyl-HSL-degrading activity of AttM were evident in the presence of wounded tissues; however, in unwounded Plant Tumors, the QS-regulated functions were weakly affected in an attM mutant. By contrast, we observed that attM conferred a selective advantage in the course of colonization of Plant Tumors. Finally, polymerase chain reaction survey of genes attM and aiiB showed that they were not strictly conserved in the genus Agrobacterium. This work proved that the lactonases AttM and AiiB are regulated by different Plant signals and are implicated in different functions in the course of the A. tumefaciens C58-host interaction.
Almudena Gonzalez-mula - One of the best experts on this subject based on the ideXlab platform.
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The biotroph Agrobacterium tumefaciens thrives in Tumors by exploiting a wide spectrum of Plant host metabolites
New Phytologist, 2019Co-Authors: Almudena Gonzalez-mula, Joy Lachat, Léo Mathias, Delphine Naquin, Florian Lamouche, Peter Mergaert, Denis FaureAbstract:Agrobacterium tumefaciens is a niche-constructing biotroph that exploits host Plant metabolites. We combined metabolomics, transposon-sequencing (Tn-seq), transcriptomics and reverse genetics to characterize A. tumefaciens pathways involved in the exploitation of resources from the Solanum lycopersicum host Plant. Metabolomics of healthy stems and Plant Tumors revealed the common (e.g., sucrose, glutamate) and enriched (e.g., opines, GABA, GHB, pyruvate) metabolites that A. tumefaciens could use as nutrients. Transposon-sequencing and transcriptomics pinpointed the genes that are crucial and/or up-regulated when the pathogen grew on either sucrose (pgi, kdgA, pycA, cisY) or GHB (blcAB, pckA, eno, gpsA) as a carbon source. While sucrose assimilation involved the Entner-Doudoroff and tricarboxylic acid (TCA) pathways, GHB degradation required the blc genes, tricarboxylic acid cycle and gluconeogenesis. The tumor-enriched metabolite pyruvate is at the node connecting these pathways. Using reverse genetics, we showed that the blc, pckA and pycA loci were important for aggressiveness (tumor weight), proliferation (bacterial charge) and/or fitness (competition between the constructed mutants and wild-type) of A. tumefaciens in Plant Tumors. This work highlighted how a biotroph mobilizes its central metabolism for exploiting a wide diversity of resources in Plant host. It further shows the complementarity of functional genome-wide scans by transcriptomics and transposon-sequencing to decipher the lifestyle of a Plant pathogen. This article is protected by copyright. All rights reserved.
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Integrative and deconvolution omics approaches to uncover the Agrobacterium tumefaciens lifestyle in Plant Tumors
Plant Signaling and Behavior, 2019Co-Authors: Almudena Gonzalez-mula, Marta Torres, Denis FaureAbstract:Agrobacterium tumefaciens is a Plant pathogen which provokes galls on roots and stems (crown-gall disease) and colonizes them. Two approaches combining omics were used to decipher the lifestyle of A. tumefaciens in Plant Tumors: an integrative approach when omics were used on A. tumefaciens cells collected from Plant Tumors, a deconvolution approach when omics were used on A. tumefaciens cells exploiting a single tumor metabolite in pure culture assay. This addendum highlights some recent results on the biotroph lifestyle of A. tumefaciens in Plant Tumors.
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The Plant GABA signaling downregulates horizontal transfer of the Agrobacterium tumefaciens virulence plasmid.
New Phytologist, 2016Co-Authors: Julien Lang, Almudena Gonzalez-mula, Ludivine Taconnat, Gilles Clement, Denis FaureAbstract:In the tumor-inducing (Ti) Agrobacterium tumefaciens, quorum sensing activates the horizontal transfer of the virulent Ti plasmid. In pure culture, this process can be impaired by the A. tumefaciens BlcC lactonase, whose expression is induced by gamma-aminobutyrate (GABA). It was therefore hypothesized that host GABA content might modulate quorum sensing and virulence gene dissemination during A. tumefaciens infection. We examined GABA metabolism and transport in Arabidopsis thaliana Tumors combining transcriptomic, metabolomic and histological approaches. In addition, using genetically modified Plants and bacteria, we evaluated the impact of Plant host GABA content on Ti plasmid dissemination. The results showed that GABA and free proline, which acts as an antagonist of GABA uptake in A. tumefaciens, accumulated in wild-type Tumors relative to uninfected Plant tissues. Moreover, comparisons of Tumors induced on Col-0 and her1 Plants showed that the increase in the Plant GABA : proline ratio was associated with both the upregulated expression of the blcC gene and the decreased dissemination of Ti plasmid in tumor-colonizing A. tumefaciens populations. This work demonstrates experimentally that the variation in the GABA content in Plant Tumors can interfere with the dissemination of A. tumefaciens Ti plasmids, and therefore highlights Plant GABA content as an important trait in the struggle against pathogenic bacteria.
Reiko Akakura - One of the best experts on this subject based on the ideXlab platform.
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Constitutive Mutations of the OccR Regulatory Protein Affect DNA Bending in Response to Metabolites Released from Plant Tumors
The Journal of biological chemistry, 2001Co-Authors: Reiko Akakura, Stephen C. WinansAbstract:OccR is a LysR-type transcriptional regulator of Agrobacterium tumefaciens that positively regulates the octopine catabolism operon of the Ti plasmid and is also an autorepressor. Positive control of the occ genes occurs in response to octopine, a nutrient released from crown gall Tumors. OccR binds to a site upstream of the occQ promoter in the presence and absence of octopine. Octopine causes prebound OccR to undergo a conformational change at the DNA binding site that causes changes in footprint length and DNA bending. To determine the roles of these conformational changes in transcriptional activation, we isolated 22 OccR mutants that were able to activate the occQ promoter in the absence of octopine. Thirteen of these mutants contained single amino acid substitutions, and nine contained two base pair changes resulting in two amino acid substitutions, which in most cases acted synergistically. These mutations spanned the entire length of the protein. Most of these mutant proteins in the absence of octopine displayed DNA binding and bending properties characteristic of transcriptionally active OccR-octopine complexes.
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diverse roles of agrobacterium ti plasmid borne genes in the formation and colonization of Plant Tumors
1999Co-Authors: Stephen C. Winans, Virginia S Kalogeraki, Santina Jafri, Reiko AkakuraAbstract:This chapter describes the various steps in Plant colonization, including binding of the bacteria to host cells, recognition of diffusible host-released chemical signals, and processing and transfer of oncogenic DNA (T-DNA). It includes a discussion on the utilization of tumor-released compounds called opines and on the horizontal transfer of the Ti plasmid. Species of Agrobacterium have been classified primarily by their phytopathogenic properties, which are largely due to differences in plasmid content. With respect to chromosomally encoded properties, A. rubi and A. vitis are well-defined separate taxa whereas A. tumefaciens, A. radiobacter, and A. rhizogenes are not. The most thoroughly studied A. tumefaciens strains contain either octopine-type or nopaline-type Ti plasmids. Virtually all genes found on Ti plasmids play direct or indirect roles in some aspect of crown gall tumorigenesis or tumor colonization. VirD4 is absolutely required for transfer and pilus formation. Some members of the vir regulon are not essential for tumorigenesis and may play other roles in pathogenesis. Since Ti plasmids encode both TraI and TraR, each conjugal donor takes a census of other donors rather than of recipients. It is a challenge even to speculate about the adaptive value of such a system. The observation that two opines regulate conjugation in opposite ways is equally perplexing.
Regine Kahmann - One of the best experts on this subject based on the ideXlab platform.
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guanyl nucleotide exchange factor sql2 and ras2 regulate filamentous growth in ustilago maydis
Eukaryotic Cell, 2003Co-Authors: Philip Muller, Jorg D Katzenberger, Gabriel Loubradou, Regine KahmannAbstract:The cyclic AMP (cAMP)-signaling pathway regulates cell morphology and plays a crucial role during pathogenic development of the Plant-pathogenic fungus Ustilago maydis. Strains lacking components of this signaling pathway, such as the Gα-subunit Gpa3 or the adenylyl cyclase Uac1, are nonpathogenic and grow filamentously. On the other hand, strains exhibiting an activated cAMP pathway due to a dominant-active allele of gpa3 display a glossy colony phenotype and are unable to proliferate in Plant Tumors. Here we present the identification of sql2 as a suppressor of the glossy colony phenotype of a gpa3Q206L strain. sql2 encodes a protein with similarity to CDC25-like guanine nucleotide exchange factors, which are known to act on Ras proteins. Overexpression of sql2 leads to filamentous growth that cannot be suppressed by exogenous cAMP, suggesting that Sql2 does not act upstream of Uac1. To gain more insight in signaling processes regulated by Sql2, we isolated two genes encoding Ras proteins. Expression of dominant active alleles of ras1 and ras2 showed that Ras2 induces filamentous growth while Ras1 does not affect cell morphology but elevates pheromone gene expression. These results indicate that Ras1 and Ras2 fulfill different functions in U. maydis. Moreover, observed similarities between the filaments induced by sql2 and ras2 suggest that Sql2 is an activator of Ras2. Interestingly, sql2 deletion mutants are affected in pathogenic development but not in mating, indicating a specific function of sql2 during pathogenesis.
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Activation of the cAMP pathway in Ustilago maydis reduces fungal proliferation and teliospore formation in Plant Tumors.
Molecular plant-microbe interactions : MPMI, 2000Co-Authors: Julia Krüger, Gabriel Loubradou, Michael Feldbrügge, Gerhard Wanner, Erika Regenfelder, Regine KahmannAbstract:In the corn smut fungus Ustilago maydis, mating of two haploid sporidia is a prerequisite for subsequent colonization of the host. Cyclic AMP (cAMP) and pheromone signals have been implicated in this developmental program. The cAMP pathway is also needed for subsequent fungal development in Planta, as null mutants in any component of the pathway fail to form Tumors. Here we show that moderate activation of the pathway conferred either by mutation in the Gα subunit or by mutation in the regulatory subunit of the protein kinase A influences tumor morphology. In the resulting Tumors, the amount of fungal material is drastically reduced and fungal development is arrested at the stage of sporogenic hyphae. We conclude that tight regulation of the cAMP pathway is crucial for fungal development within the Plant but does not interfere with the tumor induction process.