Group Lars Voll

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Molecular Interactions

Plant metabolism is under complex developmental and environmental control ensuring coordinated interaction between (photo)autotrophic source leaves and sink tissues, like developing leaves, roots, flowers and storage organs. The metabolic adjustment between source and sink organs is a prerequisite for normal plant growth and propagation and, hence, a major determinant of crop yield. However, source leaf productivity and source-sink interactions are challenged by both, biotic and abiotic stresses.

Identification of metabolic compatibility factors in plant-fungal interactions

Phytopathogenic fungi have evolved sophisticated strategies to alter sink-to-source relations of their host plants and to concomitantly suppress host defence responses. Biotrophic and hemibiotrophic fungi, which depend on living host cells at least during part of their life cycle, manipulate plant metabolism at the infection site such that carbon and nitrogen assimilate allocation and production are altered in favour of the invader. Understanding the underlying mechanisms of how fungal pathogens achieve the reprogramming of host metabolism should enable molecular breeding of crop plants with improved fungal resistance and with increased harvest index - i.e. when mechanistic insight on how assimilate production and allocation are manipulated by the pathogen is being utilized to increase crop yield. Knowledge about fungal effector proteins involved in host reprogramming and on the corresponding host targets is about to emerge, but yet largely unknown. Likewise, information on the role of particular metabolic processes for compatibility in plant-fungal interactions is scarce.

The major objective of the Metabolic Interactions group is to elucidate how metabolic processes are involved in determining fungal pathogenicity and host susceptibility and to identify molecular and metabolic key players in both, host and pathogen. Employing mutants and transgenics of both interaction partners, we study the importance of particular steps in primary carbon and nitrogen metabolism for i) pathogen invasion and ii) host defence effectiveness. Analyzing physiological aspects in the interactions on the systems level is an integral part of our approach.

Figure 1.
left: Leaf phenotype 8 days after infection of maize plantlets with the biotrophic phytopathogenic fungus Ustilago maydis.   Photograph by R.J.Horst.
right: Leaf phenotype of Arabidopsis plants 4 days after infection with the hemibiotrophic fungus Colletotrichum higginsianum (right plant) compared to non-infected control (left plant).   Photograph by T. Engelsdorf.

As part of the DFG-Forschergruppe 666 (FOR666), several plant-fungal interactions are studied, most of which involve cereals.

(A)   Biotrophic fungi:

  • Blumeria graminis f.sp. hordei on barley
  • Ustilago maydis on maize (Figures 1 & 3)

(B)   Hemibiotrophic fungi:

  • Colletotrichum graminicola on maize (Figure 3)
  • Colletotrichum higginsianum on Arabidopsis thaliana (Figure 1)

The major drivers for the generation and validation of our scientific hypotheses are

(a)   Transcriptome analysis on Agilent custom microarrays - supported by the Transcriptomics group.

(b)   Metabolome analyses by

  • 13C und 15N stable isotope labelling kinetics,
  • metabolic fingerprinting and
  • targeted metabolomics,

employing HPLC, IC, HPLC-MS/MS, IC-MS/MS, GC-MS and spectrophotometric assays, which is performed in cooperation with the Bioanalytics group.
To assess effects on the whole plant and whole leaf level, physiological approaches are being undertaken that include

(c)   a combined IRGA-Imaging-PAM system (see Figure 2 below) for the simultaneous measurement of photosynthetic gas exchange (IRGA) coupled to spatial chlorophyll fluorescence imaging (PAM).

 

Figure 2.   Setup of the Walz GFS-3000 combined infrared gas analyzer (IRGA)-chlorophyll imaging system.

As an example, chlorophyll imaging analysis of maize leaf photosynthesis after infection with the biotrophic pathogen Ustilago maydis and the hemibiotrophic fungal pathogen Colletotrichum graminicola reveals spatially restricted changes relative to control leaves (Figure 3).

Figure 3.   Chlorophyll fluorescence imaging of infected maize leaves.
left: Chlorophyll fluorescence imaging analysis of maize leaves infected with the biotrophic fungus U. maydis (right) and mock control leaves (left). The maximum photosystem II quantum yield Fv/Fm (top), non-photochemical quenching. i.e. regulated energy dissipation, qN (middle) and non-regulated energy dissipation Y(NO) (bottom) are shown in false color images with the scale ranging from 0 (black) to 1 (purple), as indicated by the color bar at the bottom.   Data by R.J. Horst.
right: Chlorophyll imaging of a maize leaf infected with the fungal hemibiotroph C. graminicola. The photosystem II quantum efficiency (ΦPSII - top) and the maximum photosystem II quantum yield Fv/Fm (middle) are shown in false color images with the scale ranging from 0 (black) to 1 (purple), as indicated by the color bar at the bottom. For comparison, an photograph of the same leaf is displayed in the bottom panel.   Data by A.M. Koszucka.

The influence of the foliar antioxidative network in abiotic stress tolerance

Another interest of the Metabolic Interactions group concerns the response of plants to abiotic stress. We employ various transgenic plants with an altered antioxidative system to learn about the role of particular antioxidants in stress tolerance and to get insight about the interaction of the different major antioxidants like ascorbate (Vitamin C), glutathione and tocochromanols (Vitamin E) in the foliar antioxidative network.

Tocochromanols are amphiphilic antioxidants abundant in thylakoids, where the photosynthetic electron transport chain operates. During the light reactions of photosynthesis, reactive oxygen species (ROS) like superoxide anions (O2-) and singlet oxygen (1O2) are generated as side products. Tocochromanols quench ROS at the site of production, thereby ameliorating the load on the soluble antioxidant system that is dominated by the ascorbate-glutathione cycle.

Most dicot plant species accumulate α-tocopherol in leaves while the seed Vitamin E pool is dominated by γ-tocopherol. Employing i) transgenic tobacco plants devoid of tocopherols and ii) transgenics, in which γ-tocopherol substitutes for α-tocopherol in leaves, our studies indicate that γ-tocopherol is more potent in conferring elevated tolerance towards several oxidative stress scenarios in leaves (as exemplified in Figure 4).

Figure 4.   Effect of sorbitol-induced oxidative stress on axenically grown tobacco plantlets after four weeks of challenge. Compared to wild type SNN (with 95% foliar α-tocopherol) or HPT transgenic lines (2% residual tocopherol in leaves), the γ-TMT transgenic lines (with 95% foliar γ-tocopherol instead of α-tocopherol) are less sensitive to sorbitol. In the γ-TMT transgenics, γ-tocopherol methyl transferase (γ-TMT) was silenced by overexpression of a γ-TMT-specific hairpin construct and in the HPT transgenics, homogentisate phytyltransferase (HPT) was post-transcriptionally silenced by the same molecular technique.   Data by P. Hennig and A.R. Abbasi.