Regulation of PIN-FORMED-mediated auxin transport and the role of auxin and cytokinin responses in plant-nematode interactions

The phytohormone auxin is a major determinant of plant growth and development. Many aspects of its action depend on the formation of local maxima or gradients within tissues. PIN-FORMED (PIN) proteins facilitate auxin efflux from cells and, by their dynamic polar localization, provide a basis for its directional transport. PIN-dependent auxin transport is regulated by several members of the plant specific AGCVIII kinase family. Phosphorylation of the central serine residues in three conserved TPRXS(N/S) motifs by PIONOID (PID) is crucial for polar targeting of PINs. The D6 protein kinases (D6PKs) were also shown to be involved in the control of polar auxin transport and are functionally linked to PIN1 and PIN3, however they do not interfere with PIN subcellular localization. In this work, the X. laevis oocyte expression system was used to study the impact of D6PK and other representative members of the AGCVIII kinase family on transport activity of PIN1 and PIN3. An assay was established that allowed monitoring auxin efflux after direct injection of oocytes with radiolabeled substrate. It could be shown that co-expression of PIN1 and PIN3 proteins with D6PK or PID results in phosphorylation of PINs and in enhanced auxin efflux. Contrarily, expression of PIN proteins alone did not cause measurable auxin efflux when compared to water-injected control oocytes. Based on the observation that a kinase-inactive version of D6PK could no longer stimulate PIN-mediated auxin efflux, it was concluded that phosphorylation is essential for PIN function. Activation of PIN1 and PIN3 could be achieved by co-expression of D6PK and PID, but not two other members of the AGCVIII kinase family, PHOT1 and UNC, demonstrating the specificity of the interaction between PINs and D6PK / PID. Analyses of different mutants lead to the identification of phosphorylation target sites that are essential for D6PK / PID – mediated PIN1 transport activity. A serine to alanine substitution at position 271 caused decreased auxin efflux from PIN1S271A / D6PK co-expressing oocytes, but had no effect on PID mediated activation, indicating that S271 is important for D6PK function, while PID acts independently of S271, i.e. vy phosphorylation of different residues. Consistently, an opposite effect was observed in a PIN1 S231A/S252A/S290A triple mutant with alanines replacing the central serines in the TPRXS(N/S) motifs. The different impacts of the S271A and the S231A/S252A/S290A mutations on D6PK and PID function suggested that activation of PIN1 by D6PK is mediated preferentially via phosphorylation of S271A whereas PID favors the central serines in the TPRXS(N/S) motifs, thereby regulating polarity as well as activity. While both PIN1S271A as well as the triple mutant PIN1 S231A/S252A/S290A could still mediate enhanced auxin efflux from oocytes when co-expressed with D6PK or PID, although to a varying extent, transport activity was almost completely lost in a combined quadruple mutant PIN1 S231A/S252A/S271A/S290A. The results obtained from the analysis of PIN1 mutants suggest that the main phosphorylation sites that contribute to PIN1 activatability by D6PK and PID have been identified and include S271 as well as S231/S252/S290. However, it was clearly demonstrated that not all of the sites have to be phosphorylated at the same time to confer transport activity to PIN1. In fact, phosphorylation of either S271 or one or more of the S231/S252/S290 sites is sufficient for transport activity under the given experimental conditions. PIN3 generally exhibited higher transport rates than PIN1 when co-expressed with an activating kinase, probably reflecting its function in processes that involve rapid polar redistribution of auxin. A serine to alanine exchange at position 262 of PIN3 which corresponds to S271 in PIN1 had no effect on the activation by either D6PK or PID when compare to wild type PIN3 indicating that different amino acid residues are important for PIN3 activity. In summary, the results obtained in the first part of this work provide evidence that the transport activity of the auxin efflux carriers PIN1 and PIN3 is controlled by the AGCVIII kinases D6PK and PID and depends on phosphorylation of specific amino acid residues, thereby giving important insights into the biochemical mechanisms that control PIN protein function. In the second part of the thesis, the role of phytohormone responses in plant-nematode interactions was studied. Plant parasitic nematodes are destructive pathogens with a broad host range and cause enormous yield losses each year. The sedentary endoparasitic nematodes are divided into two groups, the root knot nematodes (RKN) and the cyst nematodes (CN). The free living juveniles of RKN and CN enter their host plant’s roots and induce the formation of highly specialized feeding sites termed giant cells or syncytia, respectively, in the vascular cylinder. From these feeding sites, all the nutrients required for growth and development of the nematodes are withdrawn. Giant cells and syncytia are functionally equivalent and represent strong terminal sink tissues. However they differ in their genesis and in the way how they are loaded with nutrients. While syncytia are connected to the surrounding vasculature by plasmodesmata, giant cells are symplastically isolated and nutrient uptake occurs via membrane dependent processes. Both syncytia and giant cells are enclosed in vascular tissue which is formed de novo and is required for the transport of nutrients towards the feeding sites. The unloading phloem that is induced around syncytia consists of sieve elements and companion cells with the ratio shifted towards an excess of sieve elements. Giant cells on the other hand are surrounded by a unique phloem tissue that is built up of nucleate sieve elements whereas companion cells are absent. It was shown before that both CN and RKN secrete auxin- and cytokinin-like compounds and that auxin plays an important role in the establishment of the feeding sites. However, nothing was known about how the vascularization of syncytia and giant cells is controlled and whether phytohormones play a role in this process. Therefore auxin and cytokinin responses were monitored in nematode infected Arabidopsis roots using the synthetic hormone responsive promoters DR5 and TCS, respectively, fused to the coding sequence for ER-localized green fluorescent protein (GFP). GFP fluorescence was recorded and immunohistochemical experiments were performed in order to identify the cell types within the nematode induced tissues that responded to the phytohormones. This provided insights into a possible function of phytohormone dependent processes in feeding site vascularization. In uninfected differentiated parts of control roots a constitutive auxin response was found in companion cells, but no response to cytokinin mediated by B-type response regulators was observed. Neither giant cells nor syncytia themselves responded to auxin or cytokinin at the timepoints of the infection cycle that were analyzed in this work but the response was limited to cells that surrounded the feeding sites. The observed hormone responses differed between RKN and CN-induced tissues. In developing root knots, an auxin response in cells around the giant cells preceded their subsequent differentiation into sieve elements, which were identified using a well established sieve element marker. Possibly, the auxin response primes these cells for the differentiation process. GFP signal was retained even after the responding cells had assumed sieve element identity while cells that tested positive only for GFP but not for the sieve element marker were an exception in the mature root knot. The latter observation adds further evidence to the previously described finding that companion cells are absent from fully developed root knots. Cytokinin signaling could not be observed in root knots of PTCS:ER-GFP plants which means that either cytokinin levels are low or cells are not susceptible to the signal. In contrast to the situation in root knots, the phloem around syncytia responded to both hormones. Auxin response was detected in companion cells and sieve elements while cytokinin response was limited to the sieve elements. Taking into account the distinct characteristics of the phloem tissues around syncytia and giant cells and the differential hormone responses, the ratio of cytokinin and auxin response might contribute to the identity of the de novo formed phloem, i.e. metaphloem in CN induced tissues and protophloem in RKN induced tissues