Here, we characterize a plastidial thioredoxin (TRX) isoform from Arabidopsis thaliana that defines a previously unknown branch of plastidial TRXs lying between x-and y-type TRXs and thus was named TRX z. An Arabidopsis knockout mutant of TRX z had a severe albino phenotype and was inhibited in chloroplast development. Quantitative real-time RT-PCR analysis of the mutant suggested that the expressions of genes that depend on a plastid-encoded RNA polymerase (PEP) were specifically decreased. Similar results were obtained upon virus-induced gene silencing (VIGS) of the TRX z ortholog in Nicotiana benthamiana. We found that two fructokinase-like proteins (FLN1 and FLN2), members of the pfkB-carbohydrate kinase family, were potential TRX z target proteins and identified conserved Cys residues mediating the FLN-TRX z interaction. VIGS in N. benthamiana and inducible RNA interference in Arabidopsis of FLNs also led to a repression of PEPdependent gene transcription. Remarkably, recombinant FLNs displayed no detectable sugar-phosphorylating activity, and amino acid substitutions within the predicted active site imply that the FLNs have acquired a new function, which might be regulatory rather than metabolic. We were able to show that the FLN2 redox state changes in vivo during light/dark transitions and that this change is mediated by TRX z. Taken together, our data strongly suggest an important role for TRX z and both FLNs in the regulation of PEP-dependent transcription in chloroplasts.

Since years, research on SnRK1, the major cellular energy sensor in plants, has tried to define its role in energy signalling. However, these attempts were notoriously hampered by the lethality of a complete knockout of SnRK1. Therefore, we generated an inducible amiRNA::SnRK1α2 in a snrk1α1 knock out background (snrk1α1/α2) to abolish SnRK1 activity to understand major systemic functions of SnRK1 signalling under energy deprivation triggered by extended night treatment. We analysed the in vivo phosphoproteome, proteome and metabolome and found that activation of SnRK1 is essential for repression of high energy demanding cell processes such as protein synthesis. The most abundant effect was the constitutively high phosphorylation of ribosomal protein S6 (RPS6) in the snrk1α1/α2 mutant. RPS6 is a major target of TOR signalling and its phosphorylation correlates with translation. Further evidence for an antagonistic SnRK1 and TOR crosstalk comparable to the animal system was demonstrated by the in vivo interaction of SnRK1α1 and RAPTOR1B in the cytosol and by phosphorylation of RAPTOR1B by SnRK1α1 in kinase assays. Moreover, changed levels of phosphorylation states of several chloroplastic proteins in the snrk1α1/α2 mutant indicated an unexpected link to regulation of photosynthesis, the main energy source in plants.

Little is known about the function of host factors involved in disease susceptibility. The barley (Hordeum vulgare) ROP (RHO of plants) G-protein RACB is required for full susceptibility of the leaf epidermis to invasion by the biotrophic fungus Blumeria graminis f. sp hordei. Stable transgenic knockdown of RACB reduced the ability of barley to accommodate haustoria of B. graminis in intact epidermal leaf cells and to form hairs on the root epidermis, suggesting that RACB is a common element of root hair outgrowth and ingrowth of haustoria in leaf epidermal cells. We further identified a barley MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN (MAGAP1) interacting with RACB in yeast and in planta. Fluorescent MAGAP1 decorated cortical microtubules and was recruited by activated RACB to the cell periphery. Under fungal attack, MAGAP1-labeled microtubules built a polarized network at sites of successful defense. By contrast, microtubules loosened where the fungus succeeded in penetration. Genetic evidence suggests a function of MAGAP1 in limiting susceptibility to penetration by B. graminis. Additionally, MAGAP1 influenced the polar organization of cortical microtubules. These results add to our understanding of how intact plant cells accommodate fungal infection structures and suggest that RACB and MAGAP1 might be antagonistic players in cytoskeleton organization for fungal entry.

The eukaryotic cytoskeleton is essential for structural support and intracellular transport, and is therefore a common target of animal pathogens. However, no phytopathogenic effector has yet been demonstrated to specifically target the plant cytoskeleton. Here we show that the Pseudomonas syringae type III secreted effector HopZ1a interacts with tubulin and polymerized microtubules. We demonstrate that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor phytic acid. Activated HopZ1a acetylates itself and tubulin. The conserved autoacetylation site of the YopJ / HopZ superfamily, K289, plays a critical role in both the avirulence and virulence function of HopZ1a. Furthermore, HopZ1a requires its acetyltransferase activity to cause a dramatic decrease in Arabidopsis thaliana microtubule networks, disrupt the plant secretory pathway and suppress cell wall-mediated defense. Together, this study supports the hypothesis that HopZ1a promotes virulence through cytoskeletal and secretory disruption.

Trehalose-6-phosphate (T6P) is a signaling metabolite that regulates carbon metabolism, developmental processes, and growth in plants. In Arabidopsis (Arabidopsis thaliana), T6P signaling is, at least in part, mediated through inhibition of the SNF1-related protein kinase SnRK1. To investigate the role of T6P signaling in a heterotrophic, starch-accumulating storage organ, transgenic potato (Solanum tuberosum) plants with altered T6P levels specifically in their tubers were generated. Transgenic lines with elevated T6P levels (B33-TPS, expressing Escherichia coli osmoregulatory trehalose synthesis A [OtsA], which encodes a T6P synthase) displayed reduced starch content, decreased ATP contents, and increased respiration rate diagnostic for high metabolic activity. On the other hand, lines with significantly reduced T6P (B33-TPP, expressing E. coli OtsB, which encodes a T6P phosphatase) showed accumulation of soluble carbohydrates, hexose phosphates, and ATP, no change in starch when calculated on a fresh weight basis, and a strongly reduced tuber yield. [14 C]Glucose feeding to transgenic tubers indicated that carbon partitioning between starch and soluble carbohydrates was not altered. Transcriptional profiling of B33-TPP tubers revealed that target genes of SnRK1 were strongly up-regulated and that T6P inhibited potato tuber SnRK1 activity in vitro. Among the SnRK1 target genes in B33-TPP tubers, those involved in the promotion of cell proliferation and growth were down-regulated, while an inhibitor of cell cycle progression was up-regulated. T6P-accumulating tubers were strongly delayed in sprouting, while those with reduced T6P sprouted earlier than the wild type. Early sprouting of B33-TPP tubers correlated with a reduced abscisic acid content. Collectively, our data indicate that T6P plays an important role for potato tuber growth.

The phytopathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) requires type III effector proteins (T3Es) for virulence. After translocation into the host cell, T3Es are thought to interact with components of host immunity to suppress defence responses. XopJ is a T3E protein from Xcv that interferes with plant immune responses; however, its host cellular target is unknown. Here we show that XopJ interacts with the proteasomal subunit RPT6 in yeast and in planta to inhibit proteasome activity. A C235A mutation within the catalytic triad of XopJ as well as a G2A exchange within the N-terminal myristoylation motif abolishes the ability of XopJ to inhibit the proteasome. Xcv ΔxopJ mutants are impaired in growth and display accelerated symptom development including tissue necrosis on susceptible pepper leaves. Application of the proteasome inhibitor MG132 restored the ability of the Xcv ΔxopJ to attenuate the development of leaf necrosis. The XopJ dependent delay of tissue degeneration correlates with reduced levels of salicylic acid (SA) and changes in defence- and senescence-associated gene expression. Necrosis upon infection with Xcv ΔxopJ was greatly reduced in pepper plants with reduced expression of NPR1, a central regulator of SA responses, demonstrating the involvement of SA-signalling in the development of XopJ dependent phenotypes. Our results suggest that XopJ-mediated inhibition of the proteasome interferes with SA-dependent defence response to attenuate onset of necrosis and to alter host transcription. A central role of the proteasome in plant defence is discussed.

Application of norflurazon (NF) damages plastids, induces photobleaching and represses expression of the nuclear LHCB1.2 gene encoding a light-harvesting protein. In genomes uncoupled (gun) mutants, LHCB1.2 expression is maintained in the presence of NF. The mutants gun2, gun4 and gun5 exhibit perturbations in tetrapyrrole biosynthesis, but gun1 is defective in organellar gene expression (OGE). How gun mutations affect nuclear gene expression (NGE) and why the signals elicited by the two types evoke the same response remains unknown. Here we show that the carotenoid biosynthesis inhibitors amitrole and flurochloridone can replace NF in gun assays, whereas novel tetrapyrrole pathway mutations do not provoke a gun phenotype. Changes in haem levels also do not account for LHCB1.2 derepression in NF-treated gun mutants. Pigment measurements indicated that gun mutants are not resistant to NF, but gun2, gun4 and gun5 retain low levels of lutein, as well as of neoxanthin and violaxanthin, the precursors of abscisic acid (ABA). This might explain the enhanced ABA sensitivity of gun4 and gun5 plants found in germination assays. Metabolite profiling and analyses of reactive oxygen species and cellular redox state failed to suggest a link between gun mutations and altered LHCB1.2 expression. However, in contrast to NF-treated wild-type plants, gun mutants retain to a marked extent the capability to express the plastome-encoded proteins AtpB and RbcL. This, together with the finding that application of ABA can partially restore LHCB1.2 expression in NF-treated wild-type plants, supports the view that tetrapyrrole, OGE and ABA signalling are interconnected.

The capsid protein (CP) of potyviruses is required for various steps during plant infection, such as virion assembly, cell-to-cell movement, and long-distance transport. This suggests a series of compatible interactions with putative host factors which, however, are largely unknown. By using the yeast two-hybrid system the CP from Potato virus Y (PVY) was found to interact with a novel subset of DnaJ-like proteins from tobacco,