A Cold-Shock Protein from the South Pole-Dwelling Soil Bacterium Arthrobacter sp. Confers Cold Tolerance to Rice.

Low temperature is a critical environmental factor restricting the physiology of organisms across kingdoms. In prokaryotes, cold shock induces the expression of various genes and proteins involved in cellular processes. Here, a cold-shock protein (ArCspA) from the South Pole-dwelling soil bacterium Arthrobacter sp. A2-5 was introduced into rice, a monocot model plant species. Four-week-old 35S:ArCspA transgenic rice plants grown in a cold chamber at 4 °C survived for 6 days. Cold stress significantly decreased the chlorophyll content in WT plants after 4 days compared with that in 35S:ArCspA transgenic plants. RNA-seq analysis was performed on WT and 35S:ArCspA transgenic rice with/without cold stress. GO terms such as "response to stress (GO:0006950)", "response to cold (GO:0009409)", and "response to heat (GO:0009408)" were significantly enriched among the upregulated genes in the 35S:ArCspA transgenic rice under normal conditions, even without cold-stress treatment. The expression of five cold stress-related genes, Rab16B (Os11g0454200), Rab21 (Os11g0454300), LEA22 (Os01g0702500), ABI5 (Os01 g0859300), and MAPK5 (Os03g0285800), was significantly upregulated in the transgenic rice compared with the WT rice. These results indicate that the ArCspA gene might be involved in the induction of cold-responsive genes and provide cold tolerance.

Deficiency of cold-inducible RNA-binding protein exacerbated monocrotaline-induced pulmonary artery hypertension through Caveolin1 and CAVIN1.

Cold-inducible RNA-binding protein (CIRP) was a crucial regulator in multiple diseases. However, its role in pulmonary artery hypertension (PAH) is still unknown. Here, we first established monocrotaline (MCT)-induced rat PAH model and discovered that CIRP was down-regulated predominantly in the endothelium of pulmonary artery after MCT injection. We then generated Cirp-knockout (Cirp-KO) rats, which manifested severer PAH with exacerbated endothelium damage in response to MCT. Subsequently, Caveolin1 (Cav1) and Cavin1 were identified as downstream targets of CIRP in MCT-induced PAH, and the decreased expression of these two genes aggravated the injury and apoptosis of pulmonary artery endothelium. Moreover, CIRP deficiency intensified monocrotaline pyrrole (MCTP)-induced rat pulmonary artery endothelial cells (rPAECs) injuries both in vivo and in vitro, which was counteracted by Cav1 or Cavin1 overexpression. In addition, CIRP regulated the proliferative effect of conditioned media from MCTP-treated rPAECs on rat pulmonary artery smooth muscle cells, which partially explained the exceedingly thickened pulmonary artery intimal media in Cirp-KO rats after MCT treatment. These results demonstrated that CIRP acts as a critical protective factor in MCT-induced rat PAH by directly regulating CAV1 and CAVIN1 expression, which may facilitate the development of new therapeutic targets for the intervention of PAH.

Salmonella Typhimurium encoded cold shock protein E is essential for motility and biofilm formation.

The ability of bacteria to form biofilms increases their survival under adverse environmental conditions. Biofilms have enormous medical and environmental impact; consequently, the factors that influence biofilm formation are an important area of study. In this investigation, the roles of two cold shock proteins (CSP) during biofilm formation were investigated in Salmonella Typhimurium, which is a major foodborne pathogen. Among all CSP transcripts studied, the expression of cspE (STM14_0732) was higher during biofilm growth. The cspE deletion strain (ΔcspE) did not form biofilms on a cholesterol coated glass surface; however, complementation with WT cspE, but not the F30V mutant, was able to rescue this phenotype. Transcript levels of other CSPs demonstrated up-regulation of cspA (STM14_4399) in ΔcspE. The cspA deletion strain (ΔcspA) did not affect biofilm formation; however, ΔcspEΔcspA exhibited higher biofilm formation compared to ΔcspE. Most likely, the higher cspA amounts in ΔcspE reduced biofilm formation, which was corroborated using cspA over-expression studies. Further functional studies revealed that ΔcspE and ΔcspEΔcspA exhibited slow swimming but no swarming motility. Although cspA over-expression did not affect motility, cspE complementation restored the swarming motility of ΔcspE. The transcript levels of the major genes involved in motility in ΔcspE demonstrated lower expression of the class III (fliC, motA, cheY), but not class I (flhD) or class II (fliA, fliL), flagellar regulon genes. Overall, this study has identified the interplay of two CSPs in regulating two biological processes: CspE is essential for motility in a CspA-independent manner whereas biofilm formation is CspA-dependent.

A Lin28 homologue reprograms differentiated cells to stem cells in the moss Physcomitrella patens.

Both land plants and metazoa have the capacity to reprogram differentiated cells to stem cells. Here we show that the moss Physcomitrella patens Cold-Shock Domain Protein 1 (PpCSP1) regulates reprogramming of differentiated leaf cells to chloronema apical stem cells and shares conserved domains with the induced pluripotent stem cell factor Lin28 in mammals. PpCSP1 accumulates in the reprogramming cells and is maintained throughout the reprogramming process and in the resultant stem cells. Expression of PpCSP1 is negatively regulated by its 3'-untranslated region (3'-UTR). Removal of the 3'-UTR stabilizes PpCSP1 transcripts, results in accumulation of PpCSP1 protein and enhances reprogramming. A quadruple deletion mutant of PpCSP1 and three closely related PpCSP genes exhibits attenuated reprogramming indicating that the PpCSP genes function redundantly in cellular reprogramming. Taken together, these data demonstrate a positive role of PpCSP1 in reprogramming, which is similar to the function of mammalian Lin28.

The pattern-recognition receptor CORE of Solanaceae detects bacterial cold-shock protein.

Plants and animals recognize microbial invaders by detecting microbe-associated molecular patterns (MAMPs) by cell surface receptors. Many plant species of the Solanaceae family detect the highly conserved nucleic acid binding motif RNP-1 of bacterial cold-shock proteins (CSPs), represented by the peptide csp22, as a MAMP. Here, we exploited the natural variation in csp22 perception observed between cultivated tomato (Solanum lycopersicum) and Solanum pennellii to map and identify the leucine-rich repeat (LRR) receptor kinase CORE (cold shock protein receptor) of tomato as the specific, high-affinity receptor site for csp22. Corroborating its function as a genuine receptor, heterologous expression of CORE in Arabidopsis thaliana conferred full sensitivity to csp22 and, importantly, it also rendered these plants more resistant to infection by the bacterial pathogen Pseudomonas syringae pv. tomato DC3000. Our study also confirms the biotechnological potential of enhancing plant immunity by interspecies transfer of highly effective pattern-recognition receptors such as CORE to different plant families.

Cold Shock Proteins Mediate GN with Mesangioproliferation.

DNA binding protein A (DbpA) is a member of the human cold shock domain-containing protein superfamily, with known functions in cell proliferation, differentiation, and stress responses. DbpA mediates tight junction-associated activities in tubular epithelial cells, but the function of DbpA in mesangial cells is unknown. Here, we found DbpA protein expression restricted to vascular smooth muscle cells in healthy human kidney tissue but profound induction of DbpA protein expression within the glomerular mesangial compartment in mesangioproliferative nephritis. In vitro, depletion or overexpression of DbpA using lentiviral constructs led to inhibition or promotion, respectively, of mesangial cell proliferation. Because platelet-derived growth factor B (PDGF-B) signaling has a pivotal role in mesangial cell proliferation, we examined the regulatory effect of PDGF-B on DbpA. In vitro studies of human and rat mesangial cells confirmed a stimulatory effect of PDGF-B on DbpA transcript numbers and protein levels. Additional in vivo investigations showed DbpA upregulation in experimental rat anti-Thy1.1 nephritis and murine mesangioproliferative nephritis models. To interfere with PDGF-B signaling, we injected nephritic rats with PDGF-B neutralizing aptamers or the MEK/ERK inhibitor U0126. Both interventions markedly decreased DbpA protein expression. Conversely, continuous PDGF-B infusion in healthy rats induced DbpA expression predominantly within the mesangial compartment. Taken together, these results indicate that DbpA is a novel target of PDGF-B signaling and a key mediator of mesangial cell proliferation.

Cold-inducible RNA-binding protein plays a central role in the pathogenesis of abdominal aortic aneurysm in a murine experimental model.

BACKGROUND: Cold-inducible RNA-binding protein (CIRP) is a recently identified proinflammatory cytokine. We hypothesize that CIRP is involved in the progression of abdominal aortic aneurysms (AAA) and that anti-CIRP treatment could inhibit this progression. METHODS: We investigated CIRP expression in the sera and aneurysmal tissues of human AAA patients and elastase-induced AAA rats. To further examine the role of CIRP in the development of AAA, anti-CIRP antibody (1 mg/kg) or nonimmunized control immunoglobulin (Ig)G (1 mg/kg) was injected via the caudal vein in the experimental AAA model. To further investigate the underlying mechanisms, RAW 267.4 cells were stimulated with recombinant murine CIRP (rmCIRP). RESULTS: In human AAA tissue, CIRP exhibited a 5.6-fold and 93% increase in mRNA and protein expression, respectively. In a rat AAA model, CIRP was upregulated significantly in a time-dependent manner in the serum and AAA tissue. The anti-CIRP antibody treatment significantly suppressed the dilation of experimental AAA. Simultaneously, inhibition of CIRP significantly attenuated the expression of matrix metalloproteinase (MMP)-2, MMP-9, tumor necrosis factor-α, and monocyte chemoattractant protein-1, and the number of CD68-positive macrophages in the experimental AAA tissue. In vitro, rmCIRP significantly increased MMP-9 messenger RNA expression in a dose-dependent manner by 1.2-fold, 2.9-fold, and 5.5-fold, respectively. Simultaneously, rmCIRP promoted RAW 264.7 cell migration, with an approximately 2.7-fold increase in the number of migrated cells. CONCLUSION: Our findings demonstrate that CIRP mediates experimental AAA development by promoting the inflammatory response and inducing MMP-9 expression, demonstrating its potential as a novel target for inhibiting the progression of AAA.

NbCSPR underlies age-dependent immune responses to bacterial cold shock protein in Nicotiana benthamiana.

Plants use receptor kinases (RKs) and receptor-like proteins (RLPs) as pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs) that are typical of whole classes of microbes. After ligand perception, many leucine-rich repeat (LRR)-containing PRRs interact with the LRR-RK BRI1-ASSOCIATED KINASE 1 (BAK1). BAK1 is thus expected to interact with unknown PRRs. Here, we used BAK1 as molecular bait to identify a previously unknown LRR-RLP required for the recognition of the csp22 peptide derived from bacterial cold shock protein. We established a method to identify proteins that interact with BAK1 only after csp22 treatment. BAK1 was expressed transiently in Nicotiana benthamiana and immunopurified after treatment with csp22. BAK1-associated proteins were identified by mass spectrometry. We identified several proteins including known BAK1 interactors and a previously uncharacterized LRR-RLP that we termed RECEPTOR-LIKE PROTEIN REQUIRED FOR CSP22 RESPONSIVENESS (NbCSPR). This RLP associates with BAK1 upon csp22 treatment, and NbCSPR-silenced plants are impaired in csp22-induced defense responses. NbCSPR confers resistance to bacteria in an age-dependent and flagellin-induced manner. As such, it limits bacterial growth and Agrobacterium-mediated transformation of flowering N. benthamiana plants. Transgenic expression of NbCSPR into Arabidopsis thaliana conferred responsiveness to csp22 and antibacterial resistance. Our method may be used to identify LRR-type RKs and RLPs required for PAMP perception/responsiveness, even when the active purified PAMP has not been defined.

Stress-responsive expression patterns and functional characterization of cold shock domain proteins in cabbage (Brassica rapa) under abiotic stress conditions.

Although the functional roles of cold shock domain proteins (CSDPs) have been demonstrated during the growth, development, and stress adaptation of Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and wheat (Triticum aestivum), the functions of CSDPs in other plants species, including cabbage (Brassica rapa), are largely unknown. To gain insight into the roles of CSDPs in cabbage under stress conditions, the genes encoding CSDPs in cabbage were isolated, and the functional roles of CSDPs in response to environmental stresses were analyzed. Real-time RT-PCR analysis revealed that the levels of BrCSDP transcripts increased during cold, salt, or drought stress, as well as upon ABA treatment. Among the five BrCSDP genes found in the cabbage genome, one CSDP (BRU12051), named BrCSDP3, was unique in that it is localized to the chloroplast as well as to the nucleus. Ectopic expression of BrCSDP3 in Arabidopsis resulted in accelerated seed germination and better seedling growth compared to the wild-type plants under high salt or dehydration stress conditions, and in response to ABA treatment. BrCSDP3 did not affect the splicing of intron-containing genes and processing of rRNAs in the chloroplast. BrCSDP3 had the ability to complement RNA chaperone-deficient Escherichia coli mutant cells under low temperatures as well as DNA- and RNA-melting abilities, suggesting that it possesses RNA chaperone activity. Taken together, these results suggest that BrCSDP3, harboring RNA chaperone activity, plays a role as a positive regulator in seed germination and seedling growth under stress conditions.

Arabidopsis dynamin-related protein 1E in sphingolipid-enriched plasma membrane domains is associated with the development of freezing tolerance.

The freezing tolerance of Arabidopsis thaliana is enhanced by cold acclimation, resulting in changes in the compositions and function of the plasma membrane. Here, we show that a dynamin-related protein 1E (DRP1E), which is thought to function in the vesicle trafficking pathway in cells, is related to an increase in freezing tolerance during cold acclimation. DRP1E accumulated in sphingolipid and sterol-enriched plasma membrane domains after cold acclimation. Analysis of drp1e mutants clearly showed that DRP1E is required for full development of freezing tolerance after cold acclimation. DRP1E fused with green fluorescent protein was visible as small foci that overlapped with fluorescent dye-labelled plasma membrane, providing evidence that DRP1E localizes non-uniformly in specific areas of the plasma membrane. These results suggest that DRP1E accumulates in sphingolipid and sterol-enriched plasma membrane domains and plays a role in freezing tolerance development during cold acclimation.

AtCSP1 regulates germination timing promoted by low temperature.

An Arabidopsis gene trap line (GT606), which disrupted the AtCSP1 gene, exhibited an early germination phenotype that was affected by stratification treatment. Comparative analysis of GUS expression in seeds at the early germination stage, with or without stratification, demonstrated that AtCSP1 expression was affected by cold temperature. Evaluation of germination assays with varying concentrations of ABA or NaCl revealed a reduced sensitivity of the atcsp1 mutant to both ABA and NaCl. Taken together, these data support the hypothesis that AtCSP1 affects early stages of seed germination subsequent to stratification treatment of seeds.