Regulation of Cell Type-Specific Immunity Networks in Arabidopsis Roots.

While root diseases are among the most devastating stresses in global crop production, our understanding of root immunity is still limited relative to our knowledge of immune responses in leaves. Considering that root performance is based on the concerted functions of its different cell types, we undertook a cell type-specific transcriptome analysis to identify gene networks activated in epidermis, cortex, and pericycle cells of Arabidopsis (Arabidopsis thaliana) roots challenged with two immunity elicitors, the bacterial flagellin-derived flg22 and the endogenous Pep1 peptide. Our analyses revealed distinct immunity gene networks in each cell type. To further substantiate our understanding of regulatory patterns underlying these cell type-specific immunity networks, we developed a tool to analyze paired transcription factor binding motifs in the promoters of cell type-specific genes. Our study points toward a connection between cell identity and cell type-specific immunity networks that might guide cell types in launching immune response according to the functional capabilities of each cell type.

Evaluation of a novel test design to determine uptake of chemicals by plant roots.

A new hydroponic study design to determine uptake of chemicals by plant roots was tested by (i) investigating uptake of [14C]-1,2,4-triazole by wheat plants in a ring test with ten laboratory organizations and (ii) studying uptake of ten other radiolabelled chemicals by potato, tomato or wheat plants in two laboratories. Replicate data from the ring test were used to calculate plant uptake factor (PUF) values (uptake into roots and shoots) and transpiration stream concentration factor (TSCF) values (uptake into shoots). Average PUF for 1,2,4-triazole was 0.73 (n=39, 95% confidence interval (CI): 0.64, 0.82) and the corresponding TSCF value was 1.03 (n=49, 95% CI: 0.76, 1.3). Boxplots and subsequent classification tree analysis of PUF and TSCF values showed that potential outlier values were >1.38 and were observed for PUF replicates with low biomass increase (ratio of final to initial biomass ≤1.739) and small initial biomass (≤1.55g) and for TSCF replicates with an increase in biomass of <0.67g over a period of eight days. Considering only valid replicate data, average values of PUF and TSCF were 0.65 (n=33, 95% CI: 0.57, 0.73) and 0.64 (n=39, 95% CI: 0.58, 0.70). The additional experiments with ten chemicals and three plant species showed that uptake was low for polar substances of high molecular weight (≥394g/mol) and that TSCF values increased with log Kow values of the tested chemicals ranging from -1.54 to 1.88 (polynomial equation with R2=0.64). A cluster analysis for three of the compounds that were tested on wheat and tomato indicated that the plant uptake was mainly determined by the substance. Overall, the findings show that the hydroponic study design allows for reliable quantification of plant uptake over a range of compound/crop combinations.

The powdery mildew-resistant Arabidopsis mlo2 mlo6 mlo12 triple mutant displays altered infection phenotypes with diverse types of phytopathogens.

Arabidopsis thaliana mlo2 mlo6 mlo12 triple mutant plants exhibit complete immunity against infection by otherwise virulent obligate biotrophic powdery mildew fungi such as Golovinomyces orontii. While this phenotype is well documented, the interaction profile of the triple mutant with other microbes is underexplored and incomplete. Here, we thoroughly assessed and quantified the infection phenotypes of two independent powdery mildew-resistant triple mutant lines with a range of microbes. These microorganisms belong to three kingdoms of life, engage in diverse trophic lifestyles, and deploy different infection strategies. We found that interactions with microbes that do not directly enter leaf epidermal cells were seemingly unaltered or showed even enhanced microbial growth or symptom formation in the mlo2 mlo6 mlo12 triple mutants, as shown for Pseudomonas syringae and Fusarium oxysporum. By contrast, the mlo2 mlo6 mlo12 triple mutants exhibited reduced host cell entry rates by Colletotrichum higginsianum, a fungal pathogen showing direct penetration of leaf epidermal cells comparable to G. orontii. Together with previous findings, the results of this study strengthen the notion that mutations in genes MLO2, MLO6 and MLO12 not only restrict powdery mildew colonization, but also affect interactions with a number of other phytopathogens.

New insights into the subcellular localization of Tubby-like proteins and their participation in the Arabidopsis-Piriformospora indica interaction.

Tubby-like proteins (TLPs) have been associated with hormone signaling and responses to abiotic and biotic stress in plants. Recently, Arabidopsis thaliana TLP3 was found to translocate from the plasma membrane of cells in response to distinct abiotic stresses, thereby activating cellular signaling. In addition, several AtTLPs were demonstrated to be necessary for normal colonization of roots by the mutualistic fungus Piriformospora indica. Here, we present evidence for the involvement of another two AtTLPs in this interaction. Furthermore, we show that plasma membrane targeting of TLPs might be conserved in other plant species, although we did not find it for all members of the protein family. Finally, the position of a GFP-tag influences the localization of AtTLP3, which needs to be considered when working with TLPs.

The mutualistic fungus Piriformospora indica colonizes Arabidopsis roots by inducing an endoplasmic reticulum stress-triggered caspase-dependent cell death.

In Arabidopsis thaliana roots, the mutualistic fungus Piriformospora indica initially colonizes living cells, which die as the colonization proceeds. We aimed to clarify the molecular basis of this colonization-associated cell death. Our cytological analyses revealed endoplasmic reticulum (ER) swelling and vacuolar collapse in invaded cells, indicative of ER stress and cell death during root colonization. Consistent with this, P. indica-colonized plants were hypersensitive to the ER stress inducer tunicamycin. By clear contrast, ER stress sensors bZIP60 and bZIP28 as well as canonical markers for the ER stress response pathway, termed the unfolded protein response (UPR), were suppressed at the same time. Arabidopsis mutants compromised in caspase 1-like activity, mediated by cell death-regulating vacuolar processing enzymes (VPEs), showed reduced colonization and decreased cell death incidence. We propose a previously unreported microbial invasion strategy during which P. indica induces ER stress but inhibits the adaptive UPR. This disturbance results in a VPE/caspase 1-like-mediated cell death, which is required for the establishment of the symbiosis. Our results suggest the presence of an at least partially conserved ER stress-induced caspase-dependent cell death pathway in plants as has been reported for metazoans.

Functional sieve element protoplasts.

Sieve element (SE) protoplasts were liberated by exposing excised phloem strands of Vicia faba to cell wall-degrading enzyme mixtures. Two types of SE protoplasts were found: simple protoplasts with forisome inclusions and composite twin protoplasts-two protoplasts intermitted by a sieve plate-of which one protoplast often includes a forisome. Forisomes are giant protein inclusions of SEs in Fabaceae. Membrane integrity of SE protoplasts was tested by application of CFDA, which was sequestered in the form of carboxyfluorescein. Further evidence for membrane intactness was provided by swelling of SE protoplasts and forisome dispersion in reaction to abrupt lowering of medium osmolarity. The absence of cell wall remnants as demonstrated by negative Calcofluor White staining allowed patch-clamp studies. At negative membrane voltages, the current-voltage relations of the SE protoplasts were dominated by a weak inward-rectifying potassium channel that was active at physiological membrane voltages of the SE plasma membrane. This channel had electrical properties that are reminiscent of those of the AKT2/3 channel family, localized in phloem cells of Arabidopsis (Arabidopsis thaliana). All in all, SE protoplasts promise to be a powerful tool in studying the membrane biology of SEs with inherent implications for the understanding of long-distance transport and signaling.