The essential role of fungal peroxisomes in plant infection.

Several filamentous fungi are ecologically and economically important plant pathogens that infect a broad variety of crops. They cause high annual yield losses and contaminate seeds and fruits with mycotoxins. Not only powerful infection structures and detrimental toxins, but also cell organelles, such as peroxisomes, play important roles in plant infection. In this review, we summarize recent research results that revealed novel peroxisomal functions of filamentous fungi and highlight the importance of peroxisomes for infection of host plants. Central for fungal virulence are two primary metabolic pathways, fatty acid ß-oxidation and the glyoxylate cycle, both of which are required to produce energy, acetyl-CoA, and carbohydrates. These are ultimately needed for the synthesis of cell wall polymers and for turgor generation in infection structures. Most novel results stem from different routes of secondary metabolism and demonstrate that peroxisomes produce important precursors and house various enzymes needed for toxin production and melanization of appressoria. All these peroxisomal functions in fungal virulence might represent elegant targets for improved crop protection.

New guidelines for fluorophore application in peroxisome targeting analyses in transient plant expression systems.

Peroxisome research has been revolutionized by proteome studies combined with in vivo subcellular targeting analyses. Yellow and cyan fluorescent protein (YFP and CFP) are the classical fluorophores of plant peroxisome research. In the new transient expression system of Arabidopsis seedlings co-cultivated with Agrobacterium we detected the YFP fusion of one candidate protein in peroxisomes, but only upon co-transformation with the peroxisome marker, CFP-PTS1. The data suggested that the YFP fusion was directed to peroxisomes due to its weak heterodimerization ability with CFP-PTS1, allowing piggy-back import into peroxisomes. Indeed, if co-expressed with monomeric Cerulean-PTS1 (mCer-PTS1), the YFP fusion was no longer matrix localized. We systematically investigated the occurrence and extent of dimerization-based piggy-back import for different fluorophore combinations in five major transient plant expression systems. In Arabidopsis seedlings and tobacco leaves both untagged YFP and monomeric Venus were imported into peroxisomes if co-expressed with CFP-PTS1 but not with mCer-PTS1. By contrast, piggy-back import of cytosolic proteins was not observed in Arabidopsis and tobacco protoplasts or in onion epidermal cells for any fluorophore combination at any time point. Based on these important results we formulate new guidelines for fluorophore usage and experimental design to guarantee reliable identification of novel plant peroxisomal proteins.

Glucanocellulosic ethanol: the undiscovered biofuel potential in energy crops and marine biomass.

Converting biomass to biofuels is a key strategy in substituting fossil fuels to mitigate climate change. Conventional strategies to convert lignocellulosic biomass to ethanol address the fermentation of cellulose-derived glucose. Here we used super-resolution fluorescence microscopy to uncover the nanoscale structure of cell walls in the energy crops maize and Miscanthus where the typical polymer cellulose forms an unconventional layered architecture with the atypical (1, 3)-ß-glucan polymer callose. This raised the question about an unused potential of (1, 3)-ß-glucan in the fermentation of lignocellulosic biomass. Engineering biomass conversion for optimized (1, 3)-ß-glucan utilization, we increased the ethanol yield from both energy crops. The generation of transgenic Miscanthus lines with an elevated (1, 3)-ß-glucan content further increased ethanol yield providing a new strategy in energy crop breeding. Applying the (1, 3)-ß-glucan-optimized conversion method on marine biomass from brown macroalgae with a naturally high (1, 3)-ß-glucan content, we not only substantially increased ethanol yield but also demonstrated an effective co-fermentation of plant and marine biomass. This opens new perspectives in combining different kinds of feedstock for sustainable and efficient biofuel production, especially in coastal regions.

Simple preparation of plant epidermal tissue for laser microdissection and downstream quantitative proteome and carbohydrate analysis.

The outwardly directed cell wall and associated plasma membrane of epidermal cells represent the first layers of plant defense against intruding pathogens. Cell wall modifications and the formation of defense structures at sites of attempted pathogen penetration are decisive for plant defense. A precise isolation of these stress-induced structures would allow a specific analysis of regulatory mechanism and cell wall adaption. However, methods for large-scale epidermal tissue preparation from the model plant Arabidopsis thaliana, which would allow proteome and cell wall analysis of complete, laser-microdissected epidermal defense structures, have not been provided. We developed the adhesive tape - liquid cover glass technique (ACT) for simple leaf epidermis preparation from A. thaliana, which is also applicable on grass leaves. This method is compatible with subsequent staining techniques to visualize stress-related cell wall structures, which were precisely isolated from the epidermal tissue layer by laser microdissection (LM) coupled to laser pressure catapulting. We successfully demonstrated that these specific epidermal tissue samples could be used for quantitative downstream proteome and cell wall analysis. The development of the ACT for simple leaf epidermis preparation and the compatibility to LM and downstream quantitative analysis opens new possibilities in the precise examination of stress- and pathogen-related cell wall structures in epidermal cells. Because the developed tissue processing is also applicable on A. thaliana, well-established, model pathosystems that include the interaction with powdery mildews can be studied to determine principal regulatory mechanisms in plant-microbe interaction with their potential outreach into crop breeding.

Secreted fungal effector lipase releases free fatty acids to inhibit innate immunity-related callose formation during wheat head infection.

The deposition of the (1,3)-ß-glucan cell wall polymer callose at sites of attempted penetration is a common plant defense response to intruding pathogens and part of the plant's innate immunity. Infection of the Fusarium graminearum disruption mutant Δfgl1, which lacks the effector lipase FGL1, is restricted to inoculated wheat (Triticum aestivum) spikelets, whereas the wild-type strain colonized the whole wheat spike. Our studies here were aimed at analyzing the role of FGL1 in establishing full F. graminearum virulence. Confocal laser-scanning microscopy revealed that the Δfgl1 mutant strongly induced the deposition of spot-like callose patches in vascular bundles of directly inoculated spikelets, while these callose deposits were not observed in infections by the wild type. Elevated concentrations of the polyunsaturated free fatty acids (FFAs) linoleic and α-linolenic acid, which we detected in F. graminearum wild type-infected wheat spike tissue compared with Δfgl1-infected tissue, provided clear evidence for a suggested function of FGL1 in suppressing callose biosynthesis. These FFAs not only inhibited plant callose biosynthesis in vitro and in planta but also partially restored virulence to the Δfgl1 mutant when applied during infection of wheat spikelets. Additional FFA analysis confirmed that the purified effector lipase FGL1 was sufficient to release linoleic and α-linolenic acids from wheat spike tissue. We concluded that these two FFAs have a major function in the suppression of the innate immunity-related callose biosynthesis and, hence, the progress of F. graminearum wheat infection.

Ectoparasitic growth of Magnaporthe on barley triggers expression of the putative barley wax biosynthesis gene CYP96B22 which is involved in penetration resistance.

BACKGROUND: Head blast caused by the fungal plant pathogen Magnaporthe oryzae is an upcoming threat for wheat and barley cultivation. We investigated the nonhost response of barley to an isolate of the Magnaporthe species complex which is pathogenic on Pennisetum spp. as a potential source for novel resistance traits. RESULTS: Array experiments identified a barley gene encoding a putative cytochrome P450 monooxygenase whose transcripts accumulate to a higher concentration in the nonhost as compared to the host interaction. The gene clusters within the CYP96 clade of the P450 plant gene family and is designated as CYP96B22. Expression of CYP96B22 was triggered during the ectoparasitic growth of the pathogen on the outside of the leaf. Usage of a fungicidal treatment and a Magnaporthe mutant confirmed that penetration was not necessary for this early activation of CYP96B22. Transcriptional silencing of CYP96B22 using Barley stripe mosaic virus led to a decrease in penetration resistance of barley plants to Magnaporthe host and nonhost isolates. This phenotype seems to be specific for the barley-Magnaporthe interaction, since penetration of the adapted barley powdery mildew fungus was not altered in similarly treated plants. CONCLUSION: Taken together our results suggest a cross-talk between barley and Magnaporthe isolates across the plant surface. Since members of the plant CYP96 family are known to be involved in synthesis of epicuticular waxes, these substances or their derivatives might act as signal components. We propose a functional overlap of CYP96B22 in the execution of penetration resistance during basal and nonhost resistance of barley against different Magnaporthe species.

Resistance of callose synthase activity to free fatty acid inhibition as an indicator of Fusarium head blight resistance in wheat.

The fungal pathogen Fusarium graminearum is the causal agent of Fusarium head blight (FHB); a devastating crop disease resulting in heavy yield losses and grain contamination with mycotoxins. We recently showed that the secreted lipase FGL1, a virulence factor of F. graminearum, targets plant defense-related callose biosynthesis during wheat head infection. This effector-like function is based on a FGL1-mediated release of polyunsaturated free fatty acids (FFA) that can inhibit callose synthase activity. The importance of FGL1 in successful wheat head colonization was demonstrated in FGL1 disruption mutants (Δfgl1), where infection was restricted to directly inoculated spikelets and accompanied by strong callose deposition in the spikelet's phloem. The application of polyunsaturated FFA to Δfgl1-infected spikelets prevented callose deposition in the phloem and partially restored wheat head colonization.   The comparative analysis of 3 wheat cultivars revealed that the level of resistance to FHB correlated with resistance to FFA-dependent inhibition of callose biosynthesis. Therefore, resistance of callose biosynthesis to FFA inhibition might be used as marker and/or direct target in the breeding of FHB-resistant wheat cultivars.

Elevated early callose deposition results in complete penetration resistance to powdery mildew in Arabidopsis.

A common response by plants to fungal attack is deposition of callose, a (1,3)-ß-glucan polymer, in the form of cell wall thickenings called papillae, at site of wall penetration. While it has been generally believed that the papillae provide a structural barrier to slow fungal penetration, this idea has been challenged in recent studies of Arabidopsis (Arabidopsis thaliana), where fungal resistance was found to be independent of callose deposition. To the contrary, we show that callose can strongly support penetration resistance when deposited in elevated amounts at early time points of infection. We generated transgenic Arabidopsis lines that express POWDERY MILDEW RESISTANT4 (PMR4), which encodes a stress-induced callose synthase, under the control of the constitutive 35S promoter. In these lines, we detected callose synthase activity that was four times higher than that in wild-type plants 6 h post inoculation with the virulent powdery mildew Golovinomyces cichoracearum. The callose synthase activity was correlated with enlarged callose deposits and the focal accumulation of green fluorescent protein-tagged PMR4 at sites of attempted fungal penetration. We observed similar results from infection studies with the nonadapted powdery mildew Blumeria graminis f. sp. hordei. Haustoria formation was prevented in resistant transgenic lines during both types of powdery mildew infection, and neither the salicylic acid-dependent nor jasmonate-dependent pathways were induced. We present a schematic model that highlights the differences in callose deposition between the resistant transgenic lines and the susceptible wild-type plants during compatible and incompatible interactions between Arabidopsis and powdery mildew.