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Key roles of glutamate metabolism and cuticle permeability in the multifaceted defense response of the abscisic acid deficient sitiens tomato mutant against Botrytis cinerea

Hamed Seifi (UGent)
(2013)
Author
Promoter
(UGent)
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Abstract
As the inevitable result of a ping-pong-type co-evolution, plants have devised sophisticated defense mechanisms to cope with the constant threat of pathogenic microorganisms. These mechanisms are diverse, varying from constructing physical barriers in order to mechanically halt pathogen penetration, to de novo synthesis of various anti-microbial compounds in order to chemically suppress pathogen growth in the host tissue. On the other hand, pathogens have also developed different virulence mechanisms to surpass plant defenses, which have ultimately culminated in shaping distinct invasion strategies, often categorized into three main groups of lifestyles, i.e. biotrophy, necrotrophy and hemibiotrophy. Biotrophic pathogens need living host tissue to establish their life cycle, whereas necrotrophs kill host cells first, and then feed on the dead tissue. The third group, hemibiotrophs, exhibit an early biotrophic phase followed by a necrotrophic lifestyle in later stages of infection. Moreover, necrotrophic pathogens have been further categorized into two major groups based on their host specificity/range, as host-specific necrotrophs, and broad-host range necrotrophs. Studying a model incompatible pathosystem in the present work, we have explored host resistance mechanisms which can efficiently suppress an archetypal broad-host range necrotrophic pathogen, the ascomycete Botrytis cinerea. B. cinerea (teleomorph: Botryotinia fuckeliana) is the causal agent of the gray mold disease (or Botrytis blight), attacking over 200 species worldwide. Our lab has previously shown that deficiency of the phytohormone abscisic acid (ABA) in the sitiens mutant of tomato (Solanum lycopersicum Mill. cv Moneymaker) results in increased resistance to the necrotrophic fungus B. cinerea through rapid hydrogen peroxide production in the epidermis, followed by epidermal hypersensitive response cell death and cell wall fortifications which ultimately suppress the pathogen progress. Moreover, microarray analysis revealed that in addition to groups of genes coding for pathogenesis related proteins and enzymes related to cell-wall structure being upregulated, expression of a cluster of genes involved in central carbon/nitrogen (C/N) metabolism also significantly increased in the resistant mutant. In the present work, we have initially provided a review on the reconfiguration of plant central C/N metabolism in response to different pathogenic lifestyles, and the potential roles it may play in plant defense responses. The main focus in the review has been directed at the plant glutamate metabolism (GM). Being at the interface between central carbon and nitrogen metabolism, the plant GM orchestrates critical defense-associated events related to nitrogen transportation, cellular redox regulation, and TCA cycle-dependent energy reprogramming. These modulations seem to eventually result in either ‘endurance’, i.e. maintaining the functionality of critical pathways involved in basic metabolism of the challenged cell; or ‘evasion’, i.e. disruption of cellular basic metabolism, facilitating the death process in the challenged cell. The success and failure of these ‘endurance’ or ‘evasion’-natured defenses appear to be highly dependent on the invasion style of the pathogen, i.e. biotrophy, hemibiotrophy and necrotrophy. In other words, alterations in the host glutamate metabolism in response to different pathogenic scenarios may function in two opposing ways, either backing the ongoing defense strategy, or being exploited by the pathogen to facilitate infection. In addition, we have shown that the efficiency of the previously observed HR-mediated defense response against the B.cinerea in the sitiens mutant of tomato is vitally dependent on restructuring of the central C/N metabolism. Our transcriptional, enzymatic and metabolic results revealed an influential role for the cytosolic glutamine synthetase (GS1) and the GABA-shunt in shaping a secondary line of defense in sitiens, functioning as an anti-cell death mechanism to tightly control the extent and localization of the defense-associated HR. Further genetic evidence, gained through virus induced gene silencing, and microscopic analysis confirmed the importance of the GABA-shunt and the GS1 genes in restricting the growth of the pathogen within the inoculation site in the sitiens mutant. It was also shown that exogenous application of GABA can reduce susceptibility to the pathogen in the wild-type plant, seemingly through the anti-cell death defense mechanism observed in sitiens. Collectively, these results suggest an interplay between primary amino acid metabolism and defense response during the necrotrophic sitiens-B.cinerea interaction, whereby the epidermal H2O2-mediated pathogen arrestment is crucially linked with a durable maintenance of basic metabolism in the challenged area. Having paid attention to the important role for a fast reaction in the observed resistance response in the mutant, we also explored early signaling events in the sitiens-B. cinerea interaction. In this part of the study, evidence has been presented that ABA-deficiency in sitiens results in increased cuticle permeability which is also positively correlated with disease resistance. It was then hypothesized that a permeable cuticle can facilitate faster perception of putative elicitors released from the pathogen, or from the mechanical attempt of pathogen penetration, resulting in a rapid induction of defense-associated genes. Moreover, the pectin molecule in sitiens was found to have a higher degree of methylesterification. Therefore, it was also proposed that upon challenge with B. cinerea, different types of oligosaccharides with more efficiency in elicitation may possibly be released from the altered pectin fraction of the sitiens cell wall. We could also isolate two biologically active compounds with possible elicitation-associated roles during early stages of the sitiens-B. cinerea interaction. Our analytical data may hint at the presence of some complex interspecies signaling compounds, formed as pectic dimers that are stabilized with chitosan fragments and calcium ions (termed as COS-OGAs). In conclusion, we have shown that ABA deficiency in the sitiens mutant of tomato has resulted in a multifaceted resistance response against the broad-host range necrotrophic pathogen B. cinerea, in which several layers of defenses, such as primary metabolism, secondary metabolism, cuticle permeability and potent signaling components, play critical roles.
Keywords
sitiens, Botrytis cinerea, plant-pathogen interactions, glutamate metabolism, necrotrophic pathogen, cuticle permeability, defense mechanisms

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Citation

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Chicago
Seifi, Hamed. 2013. “Key Roles of Glutamate Metabolism and Cuticle Permeability in the Multifaceted Defense Response of the Abscisic Acid Deficient Sitiens Tomato Mutant Against Botrytis Cinerea”. Ghent, Belgium: Ghent University. Faculty of Bioscience Engineering.
APA
Seifi, H. (2013). Key roles of glutamate metabolism and cuticle permeability in the multifaceted defense response of the abscisic acid deficient sitiens tomato mutant against Botrytis cinerea. Ghent University. Faculty of Bioscience Engineering, Ghent, Belgium.
Vancouver
1.
Seifi H. Key roles of glutamate metabolism and cuticle permeability in the multifaceted defense response of the abscisic acid deficient sitiens tomato mutant against Botrytis cinerea. [Ghent, Belgium]: Ghent University. Faculty of Bioscience Engineering; 2013.
MLA
Seifi, Hamed. “Key Roles of Glutamate Metabolism and Cuticle Permeability in the Multifaceted Defense Response of the Abscisic Acid Deficient Sitiens Tomato Mutant Against Botrytis Cinerea.” 2013 : n. pag. Print.
@phdthesis{3239906,
  abstract     = {As the inevitable result of a ping-pong-type co-evolution, plants have devised sophisticated defense mechanisms to cope with the constant threat of pathogenic microorganisms. These mechanisms are diverse, varying from constructing physical barriers in order to mechanically halt pathogen penetration, to de novo synthesis of various anti-microbial compounds in order to chemically suppress pathogen growth in the host tissue. On the other hand, pathogens have also developed different virulence mechanisms to surpass plant defenses, which have ultimately culminated in shaping distinct invasion strategies, often categorized into three main groups of lifestyles, i.e. biotrophy, necrotrophy and hemibiotrophy. Biotrophic pathogens need living host tissue to establish their life cycle, whereas necrotrophs kill host cells first, and then feed on the dead tissue. The third group, hemibiotrophs, exhibit an early biotrophic phase followed by a necrotrophic lifestyle in later stages of infection. Moreover, necrotrophic pathogens have been further categorized into two major groups based on their host specificity/range, as host-specific necrotrophs, and broad-host range necrotrophs. 
Studying a model incompatible pathosystem in the present work, we have explored host resistance mechanisms which can efficiently suppress an archetypal broad-host range necrotrophic pathogen, the ascomycete Botrytis cinerea. B. cinerea (teleomorph: Botryotinia fuckeliana) is the causal agent of the gray mold disease (or Botrytis blight), attacking over 200 species worldwide. Our lab has previously shown that deficiency of the phytohormone abscisic acid (ABA) in the sitiens mutant of tomato (Solanum lycopersicum  Mill. cv Moneymaker) results in increased resistance to the necrotrophic fungus B. cinerea through rapid hydrogen peroxide  production in the epidermis, followed by epidermal  hypersensitive response cell death and cell wall fortifications which ultimately suppress the pathogen progress. Moreover, microarray analysis revealed that in addition to groups of genes coding for pathogenesis related proteins and enzymes related to cell-wall structure being upregulated, expression of a cluster of genes involved in central carbon/nitrogen (C/N) metabolism also significantly increased in the resistant mutant. 
In the present work, we have initially provided a review on the reconfiguration of plant central C/N metabolism in response to different pathogenic lifestyles, and the potential roles it may play in plant defense responses. The main focus in the review has been directed at the plant glutamate metabolism (GM). Being at the interface between central carbon and nitrogen metabolism, the plant GM orchestrates critical defense-associated events related to nitrogen transportation, cellular redox regulation, and TCA cycle-dependent energy reprogramming. These modulations seem to eventually result in either {\textquoteleft}endurance{\textquoteright}, i.e. maintaining the functionality of critical pathways involved in basic metabolism of the challenged cell; or {\textquoteleft}evasion{\textquoteright}, i.e. disruption of cellular basic metabolism, facilitating the death process in the challenged cell. The success and failure of these {\textquoteleft}endurance{\textquoteright} or {\textquoteleft}evasion{\textquoteright}-natured defenses appear to be highly dependent on the invasion style of the pathogen, i.e. biotrophy, hemibiotrophy and necrotrophy. In other words, alterations in the host glutamate metabolism in response to different pathogenic scenarios may function in two opposing ways, either backing the ongoing defense strategy, or being exploited by the pathogen to facilitate infection.  
In addition, we have shown that the efficiency of the previously observed HR-mediated defense response against the B.cinerea in the sitiens mutant of tomato is vitally dependent on restructuring of the central C/N metabolism. Our transcriptional, enzymatic and metabolic results revealed an influential role for the cytosolic glutamine synthetase (GS1) and the GABA-shunt in shaping a secondary line of defense in sitiens, functioning as an anti-cell death mechanism to tightly control the extent and localization of the defense-associated HR. Further genetic evidence, gained through virus induced gene silencing, and microscopic analysis confirmed the importance of the GABA-shunt and the GS1 genes in restricting the growth of the pathogen within the inoculation site in the sitiens mutant. It was also shown that exogenous application of GABA can reduce susceptibility to the pathogen in the wild-type plant, seemingly through the anti-cell death defense mechanism observed in sitiens. Collectively, these results suggest an interplay between primary amino acid metabolism and defense response during the necrotrophic sitiens-B.cinerea interaction, whereby the epidermal H2O2-mediated pathogen arrestment is crucially linked with a durable maintenance of basic metabolism in the challenged area.
Having paid attention to the important role for a fast reaction in the observed resistance response in the mutant, we also explored early signaling events in the sitiens-B. cinerea interaction. In this part of the study, evidence has been presented that ABA-deficiency in sitiens results in increased cuticle permeability which is also positively correlated with disease resistance. It was then hypothesized that a permeable cuticle can facilitate faster perception of putative elicitors released from the pathogen, or from the mechanical attempt of pathogen penetration, resulting in a rapid induction of defense-associated genes. Moreover, the pectin molecule in sitiens was found to have a higher degree of methylesterification. Therefore, it was also proposed that upon challenge with B. cinerea, different types of oligosaccharides with more efficiency in elicitation may possibly be released from the altered pectin fraction of the sitiens cell wall. We could also isolate two biologically active compounds with possible elicitation-associated roles during early stages of the sitiens-B. cinerea interaction. Our analytical data may hint at the presence of some complex interspecies signaling compounds, formed as pectic dimers that are stabilized with chitosan fragments and calcium ions (termed as COS-OGAs). 
In conclusion, we have shown that ABA deficiency in the sitiens mutant of tomato has resulted in a multifaceted resistance response against the broad-host range necrotrophic pathogen B. cinerea, in which several layers of defenses, such as primary metabolism, secondary metabolism, cuticle permeability and potent signaling components, play critical roles.},
  author       = {Seifi, Hamed},
  isbn         = {9789059896239},
  keyword      = {sitiens,Botrytis cinerea,plant-pathogen interactions,glutamate metabolism,necrotrophic pathogen,cuticle permeability,defense mechanisms},
  language     = {eng},
  pages        = {I, 191},
  publisher    = {Ghent University. Faculty of Bioscience Engineering},
  school       = {Ghent University},
  title        = {Key roles of glutamate metabolism and cuticle permeability in the multifaceted defense response of the abscisic acid deficient sitiens tomato mutant against Botrytis cinerea},
  year         = {2013},
}