Cytoplasmic Kinase Network Mediates Defense Response to Spodoptera litura in Arabidopsis.

Plants defend against folivores by responding to folivore-derived elicitors following activation of signaling cascade networks. In Arabidopsis, HAK1, a receptor-like kinase, responds to polysaccharide elicitors (Frα) that are present in oral secretions of Spodoptera litura larvae to upregulate defense genes (e.g., PDF1.2) mediated through downstream cytoplasmic kinase PBL27. Here, we explored whether other protein kinases, including CPKs and CRKs, function with PBL27 in the intracellular signaling network for anti-herbivore responses. We showed that CRK2 and CRK3 were found to interact with PBL27, but CPKs did not. Although transcripts of PDF1.2 were upregulated in leaves of wild-type Arabidopsis plants in response to mechanical damage with Frα, this failed in CRK2- and PBL27-deficient mutant plants, indicating that the CRK2/PBL27 system is predominantly responsible for the Frα-responsive transcription of PDF1.2 in S. litura-damaged plants. In addition to CRK2-phosphorylated ERF13, as shown previously, ethylene signaling in connection to CRK2-phosphorylated PBL27 was predicted to be responsible for transcriptional regulation of a gene for ethylene response factor 13 (ERF13). Taken together, these findings show that CRK2 regulates not only ERF13 phosphorylation but also PBL27-dependent de novo synthesis of ERF13, thus determining active defense traits against S. litura larvae via transcriptional regulation of PDF1.2.

ADP-Induced Conformational Transition of Human Adenylate Kinase 1 Is Triggered by Suppressing Internal Motion of α3α4 and α7α8 Fragments on the ps-ns Timescale.

Human adenylate kinase 1 (hAK1) plays a vital role in the energetic and metabolic regulation of cell life, and impaired functions of hAK1 are closely associated with many diseases. In the presence of Mg2+ ions, hAK1 in vivo can catalyze two ADP molecules into one ATP and one AMP molecule, activating the downstream AMP signaling. The ADP-binding also initiates AK1 transition from an open conformation to a closed conformation. However, how substrate binding triggers the conformational transition of hAK1 is still unclear, and the underlying molecular mechanisms remain elusive. Herein, we determined the solution structure of apo-hAK1 and its key residues for catalyzing ADP, and characterized backbone dynamics characteristics of apo-hAK1 and hAK1-Mg2+-ADP complex (holo-hAK1) using NMR relaxation experiments. We found that ADP was primarily bound to a cavity surrounded by the LID, NMP, and CORE domains of hAK1, and identified several critical residues for hAK1 catalyzing ADP including G16, G18, G20, G22, T39, G40, R44, V67, D93, G94, D140, and D141. Furthermore, we found that apo-hAK1 adopts an open conformation with significant ps-ns internal mobility, and Mg2+-ADP binding triggered conformational transition of hAK1 by suppressing the ps-ns internal motions of α3α4 in the NMP domain and α7α8 in the LID domain. Both α3α4 and α7α8 fragments became more rigid so as to fix the substrate, while the catalyzing center of hAK1 experiences promoted µs-ms conformational exchange, potentially facilitating catalysis reaction and conformational transition. Our results provide the structural basis of hAK1 catalyzing ADP into ATP and AMP, and disclose the driving force that triggers the conformational transition of hAK1, which will deepen understanding of the molecular mechanisms of hAK1 functions.

The Potassium Transporter Hak1 in Candida Albicans, Regulation and Physiological Effects at Limiting Potassium and under Acidic Conditions.

The three families of yeast plasma membrane potassium influx transporters are represented in Candida albicans: Trk, Acu, and Hak proteins. Hak transporters work as K+-H+ symporters, and the genes coding for Hak proteins are transcriptionally activated under potassium limitation. This work shows that C. albicans mutant cells lacking CaHAK1 display a severe growth impairment at limiting potassium concentrations under acidic conditions. This is the consequence of a defective capacity to transport K+, as indicated by potassium absorption experiments and by the kinetics parameters of Rb+ (K+) transport. Moreover, hak1- cells are more sensitive to the toxic cation lithium. All these phenotypes became much less robust or even disappeared at alkaline growth conditions. Finally, transcriptional studies demonstrate that the hak1- mutant, in comparison with HAK1+ cells, activates the expression of the K+/Na+ ATPase coded by CaACU1 in the presence of Na+ or in the absence of K+.

Melatonin promotes potassium deficiency tolerance by regulating HAK1 transporter and its upstream transcription factor NAC71 in wheat.

Melatonin (MT) is involved in various physiological processes and stress responses in animals and plants. However, little is known about the molecular mechanisms by which MT regulates potassium deficiency (DK) tolerance in crops. In this study, an appropriate concentration (50 µmol/L) was found to enhance the tolerance of wheat plants against DK. RNA-seq analysis showed that a total of 6253 and 5873 differentially expressed genes (DEGs) were separately identified in root and leaf tissues of the DK + MT-treated wheat plants. They functionally involved biological processes of secondary metabolite, signal transduction, and transport or catabolism. Of these, an upregulated high-affinity K transporter 1 (TaHAK1) gene was next characterized. TaHAK1 overexpression markedly enhanced the K absorption, while its transient silencing exhibited the opposite effect, suggesting its important role in MT-mediated DK tolerance. Moreover, yeast one-hybrid (Y1H) was used to screen the upstream regulators of TaHAK1 gene and the transcription factor TaNAC71 was identified. The binding between TaNAC71 and TaHAK1 promoter was evidenced by using Y1H, LUC, and EMSA assays. Transient overexpression of TaNAC71 in wheat protoplasts activated the TaHAK1 expression, whereas its transient silencing inhibited the TaHAK1 expression and aggravated the sensitivity to DK. Exogenous MT application greatly upregulated the expression of TaHAK1 in both transient overexpression and silencing systems. Our findings revealed some molecular mechanisms underlying MT-mediated DK tolerance and helped broaden its practical application in agriculture.

Regulation and activity of CaTrk1, CaAcu1 and CaHak1, the three plasma membrane potassium transporters in Candida albicans.

Wild-type cells of Candida albicans, the most common human fungal pathogen, are able to grow at very low micromolar concentrations of potassium in the external milieu. One of the reasons behind that behaviour is the existence of three different types of K+ transporters in their plasma membrane: Trk1, Acu1 and Hak1. This work shows that the transporters are very differently regulated at the transcriptional level upon exposure to saline stress, pH alterations or K+ starvation. We propose that different transporters take the lead in the diverse environmental conditions, Trk1 being the "house-keeping" one, and Acu1/Hak1 dominating upon K+ limiting conditions. Heterologous expression of the genes coding for the three transporters in a Saccharomyces cerevisiae strain lacking its endogenous potassium transporters showed that all of them mediated cation transport but with very different efficiencies. Moreover, expression of the transporters in S. cerevisiae also affected other physiological characteristics such as sodium and lithium tolerance, membrane potential or intracellular pH, being, in general, CaTrk1 the most effective in keeping these parameters close to the usual wild-type physiological levels.

Production of low-Cs+ rice plants by inactivation of the K+ transporter OsHAK1 with the CRISPR-Cas system.

The occurrence of radiocesium in food has raised sharp health concerns after nuclear accidents. Despite being present at low concentrations in contaminated soils (below µm), cesium (Cs+ ) can be taken up by crops and transported to their edible parts. This plant capacity to take up Cs+ from low concentrations has notably affected the production of rice (Oryza sativa L.) in Japan after the nuclear accident at Fukushima in 2011. Several strategies have been put into practice to reduce Cs+ content in this crop species such as contaminated soil removal or adaptation of agricultural practices, including dedicated fertilizer management, with limited impact or pernicious side-effects. Conversely, the development of biotechnological approaches aimed at reducing Cs+ accumulation in rice remain challenging. Here, we show that inactivation of the Cs+ -permeable K+ transporter OsHAK1 with the CRISPR-Cas system dramatically reduced Cs+ uptake by rice plants. Cs+ uptake in rice roots and in transformed yeast cells that expressed OsHAK1 displayed very similar kinetics parameters. In rice, Cs+ uptake is dependent on two functional properties of OsHAK1: (i) a poor capacity of this system to discriminate between Cs+ and K+ ; and (ii) a high capacity to transport Cs+ from very low external concentrations that is likely to involve an active transport mechanism. In an experiment with a Fukushima soil highly contaminated with 137 Cs+ , plants lacking OsHAK1 function displayed strikingly reduced levels of 137 Cs+ in roots and shoots. These results open stimulating perspectives to smartly produce safe food in regions contaminated by nuclear accidents.

Three Candida albicans potassium uptake systems differ in their ability to provide Saccharomyces cerevisiae trk1trk2 mutants with necessary potassium.

Yeasts usually have one or two high-affinity potassium transporters. Two complete and one interrupted gene encoding three types of putative potassium uptake system exist in Candida albicans SC5314. As high intracellular potassium is essential for many yeast cell functions, the existence of three transporters with differing transport mechanisms (Trk uniporter, Hak cation-proton symporter, Acu ATPase) may help pathogenic C. albicans cells to acquire the necessary potassium in various organs and tissues of the host. When expressed in Saccharomyces cerevisiae lacking their own potassium uptake systems, all three putative transporters were able to provide cells with the ability to grow with low amounts of potassium over a broad range of external pH. Only CaTrk1 was properly recognized and secreted to the plasma membrane. Nevertheless, even the small number of CaHak1 and mainly CaAcu1 molecules which reached the plasma membrane resulted in an improved growth of cells in low potassium concentrations, suggesting a high affinity and capacity of the transporters. A single-point mutation restored the complete CaACU1 gene, and the resulting protein not only provided cells with the necessary potassium but also improved their tolerance to toxic lithium. In contrast to its known homologues, CaAcu1 did not seem to transport sodium.