PHR2, a central regulator of phosphate signaling in rice, enhanced the expression of phosphate starvation-induced (PSI) genes and resulted in the enhancement of Pi acquisition under Pi deficiency stress. This occurred via PHR2 binding to a cis-element named the PHR1 binding sequences. However, the transcription level of PHR2 was not responsive to Pi starvation. So how is activity of transcription factor PHR2 adjusted to adapt diverse Pi status? Here, we identify an SPX family protein, Os-SPX4 (SPX4 hereafter), involving in Pi starvation signaling and acting as a negative regulator of PHR2. SPX4 is shown to be a fast turnover protein. When Pi is sufficient, through its interaction with PHR2, SPX4 inhibits the binding of PHR2 to its cis-element and reduces the targeting of PHR2 to the nucleus. However, when plants grow under Pi deficiency, the degradation of SPX4 is accelerated through the 26S proteasome pathway, thereby releasing PHR2 into the nucleus and activating the expression of PSI genes. Because the level of SPX4 is responsive to Pi concentration and SPX4 interacts with PHR2 and regulates its activity, this suggests that SPX4 senses the internal Pi concentration under diverse Pi conditions and regulates appropriate responses to maintain Pi homeostasis in plants.

In plants, sensing the levels of external and internal nutrients is essential for reprogramming the transcriptome and adapting to the fluctuating environment. Phosphate (Pi) is a key plant nutrient, and a large proportion of Pi starvation-responsive genes are under the control of PHOSPHATE STARVATION RESPONSE REGULATOR 1 (PHR1) in Arabidopsis (AtPHR1) and its homologs, such as Oryza sativa (Os)PHR2 in rice. AtPHR1 and OsPHR2 expression is not very responsive to Pi starvation, raising the question as to how plants sense changes in cellular Pi levels to activate the central regulator. SPX [named after SYG1 (suppressor of yeast gpa1), Pho81 (CDK inhibitor in yeast PHO pathway), and XPR1 (xenotropic and polytropic retrovirus receptor)] proteins that harbor only the SPX domain are reported to be involved in the negative regulation of Pi starvation responses. Here, we show that the nuclear localized SPX proteins SPX1 and SPX2 are Pi-dependent inhibitors of the activity of OsPHR2 in rice. Indeed, SPX1 and SPX2 proteins interact with PHR2 through their SPX domain, inhibiting its binding to P1BS (the PHR1-binding sequence: GNATATNC). In vivo data, as well as results from in vitro experiments using purified SPX1, SPX2, and OsPHR2 proteins, showed that SPX1 and SPX2 inhibition of OsPHR2 activity is Pi-dependent. These data provide evidence to support the involvement of SPX1 and SPX2 in the Pi-sensing mechanism in plants.SPX-domain protein | PHR2 | Pi signaling | Pi-dependent inhibition P hosphorus (P) is an essential macroelement for plant growth and development. Because of high chemical fixation, slow diffusion, and substantial fractions of organic compounds by microorganisms, phosphate (Pi) limitation is usually a constraint for crop production in cultivated soils (1). However, intensive application of P fertilizer to increase agricultural production results in higher cost and environmental pollution and aggravates the shortage of nonrenewable resources worldwide for P fertilizer production (2). Therefore, improving effective Pi use by crops to reduce agricultural dependence on heavy Pi fertilizer application is an important challenge for sustainable agricultural production.The role of Arabidopsis PHOSPHATE STARVATION RESPONSE REGULATOR 1 (AtPHR1) and its orthologs as important regulators in Pi signaling and homeostasis through binding to the PHR1-binding sequence (P1BS) is well established in plants. AtPHR1 binds as a dimer to an imperfect palindromic sequence (GNATATNC), and this DNA-binding ability is dependent on the MYB and coiled-coil (CC) domains present in AtPHR1 and related proteins (3, 4). Orthologs of AtPHR1 have also been described in rice [Oryza sativa ( The SPX domain (Pfam PF03105) is named after the suppressor of yeast gpa1 (SYG1), the yeast cyclin-dependent kinase inhibitor (PHO81), and the human xenotropic and polytropic retrovirus receptor 1 (XPR1). In yeast (Saccharomyces cerevisiae), the SPX domain forms part of the competitive dual-transporter system that prolongs preparation for starvation and faci...

In macromolecular crystallography, the rigorous detection of changed states (for example, ligand binding) is difficult unless signal is strong. Ambiguous (‘weak' or ‘noisy') density is experimentally common, since molecular states are generally only fractionally present in the crystal. Existing methodologies focus on generating maximally accurate maps whereby minor states become discernible; in practice, such map interpretation is disappointingly subjective, time-consuming and methodologically unsound. Here we report the PanDDA method, which automatically reveals clear electron density for the changed state—even from inaccurate maps—by subtracting a proportion of the confounding ‘ground state'; changed states are objectively identified from statistical analysis of density distributions. The method is completely general, implying new best practice for all changed-state studies, including the routine collection of multiple ground-state crystals. More generally, these results demonstrate: the incompleteness of atomic models; that single data sets contain insufficient information to model them fully; and that accuracy requires further map-deconvolution approaches.

Our study highlights the overactivated neddylation pathway in lung cancer development and as a promising therapeutic target.

Purpose: Innate immunity is an indispensable arm of tumor immune surveillance, and the liver is an organ with a predominance of innate immunity, where mucosal-associated invariant T (MAIT) cells are enriched. However, little is known about the phenotype, functions, and immunomodulatory role of MAIT cells in hepatocellular carcinoma (HCC). Experimental Design: The distribution, phenotype, and function of MAIT cells in patients with HCC were evaluated by both flow cytometry (FCM) and in vitro bioassays. Transcriptomic analysis of MAIT cells was also performed. Prognostic significance of tumor-infiltrating MAIT cells was validated in four independent cohorts of patients with HCC. Results: Despite their fewer densities in HCC tumor than normal liver, MAIT cells were significantly enriched in the HCC microenvironment compared with other mucosaassociated organs. Tumor-derived MAIT cells displayed a typical CCR7 À CD45RA À CD45RO þ CD95 þ effector memory phenotype with lower costimulatory and effector capabilities. Tumor-educated MAIT cells significantly upregulated inhibitory molecules like PD-1, CTLA-4, TIM-3, secreted significantly less IFNg and IL17, and produced minimal granzyme B and perforin while shifting to produce tumor-promoting cytokines like IL8. Transcriptome sequencing confirmed that tumor-derived MAIT cells were reprogrammed toward a tumor-promoting direction by downregulating genes enriched in pathways of cytokine secretion and cytolysis effector function like NFKB1 and STAT5B and by upregulating genes like IL8, CXCL12, and HAVCR2 (TIM-3). High infiltration of MAIT cells in HCC significantly correlated with an unfavorable clinical outcome, revealed by FCM, qRT-PCR, and multiplex IHC analyses, respectively. Conclusions: HCC-infiltrating MAIT cells were functionally impaired and even reprogrammed to shift away from antitumor immunity and toward a tumor-promoting direction. See related commentary by Carbone, p. 3199