Risk adjustment model for tuberculosis compared to non-tuberculosis mycobacterium or latent tuberculosis infection: Center for Personalized Precision Medicine of Tuberculosis (cPMTb) cohort database.

BACKGROUND: The Center for Personalized Precision Medicine of Tuberculosis (cPMTb) was constructed to develop personalized pharmacotherapeutic systems for tuberculosis (TB). This study aimed to introduce the cPMTb cohort and compare the distinct characteristics of patients with TB, non-tuberculosis mycobacterium (NTM) infection, or latent TB infection (LTBI). We also determined the prevalence and specific traits of polymorphisms in N-acetyltransferase-2 (NAT2) and solute carrier organic anion transporter family member 1B1 (SLCO1B1) phenotypes using this prospective multinational cohort. METHODS: Until August 2021, 964, 167, and 95 patients with TB, NTM infection, and LTBI, respectively, were included. Clinical, laboratory, and radiographic data were collected. NAT2 and SLCO1B1 phenotypes were classified by genomic DNA analysis. RESULTS: Patients with TB were older, had lower body mass index (BMI), higher diabetes rate, and higher male proportion than patients with LTBI. Patients with NTM infection were older, had lower BMI, lower diabetes rate, higher previous TB history, and higher female proportion than patients with TB. Patients with TB had the lowest albumin levels, and the prevalence of the rapid, intermediate, and slow/ultra-slow acetylator phenotypes were 39.2%, 48.1%, and 12.7%, respectively. The prevalence of rapid, intermediate, and slow/ultra-slow acetylator phenotypes were 42.0%, 44.6%, and 13.3% for NTM infection, and 42.5%, 48.3%, and 9.1% for LTBI, respectively, which did not differ significantly from TB. The prevalence of the normal, intermediate, and lower transporter SLCO1B1 phenotypes in TB, NTM, and LTBI did not differ significantly; 74.9%, 22.7%, and 2.4% in TB; 72.0%, 26.1%, and 1.9% in NTM; and 80.7%, 19.3%, and 0% in LTBI, respectively. CONCLUSIONS: Understanding disease characteristics and identifying pharmacokinetic traits are fundamental steps in optimizing treatment. Further longitudinal data are required for personalized precision medicine. TRIAL REGISTRATION: This study registered ClinicalTrials.gov NO. NCT05280886.

Glucose deprivation enhances resistance to paclitaxel via ELAVL2/4-mediated modification of glycolysis in ovarian cancer cells.

The dysregulation of glycolysis regardless of oxygen availability is one of the major characteristics of cancer cells. While the drug resistance of ovarian cancer cells has been extensively studied, the molecular mechanism of anticancer drug resistance under low-glucose conditions remains unknown. In this study, we investigated the pathway mediating drug resistance under low-glucose conditions by examining the relationship between embryonic lethal abnormal vision Drosophila homolog-like (ELAVL) protein and glycolysis-related enzymes. Ovarian cancer cells resistant to 2.5 nM paclitaxel were exposed to low-glucose media for 2 weeks, and the expression levels of ELAVL2, ELAVL4, glycolytic enzymes, and drug resistance-related proteins were elevated to levels comparable to those in cells resistant to 100 nM paclitaxel. Gene silencing of ELAVL2/4 using small interfering RNA prevented the upregulation of glycolysis-related enzymes, reduced lactate production, and sensitized 2.5 nM paclitaxel-resistant ovarian cancer cells to anticancer agents under hypoglycemic conditions. Furthermore, pharmacological inhibition of glycolytic enzymes with 2-deoxyglucose, a specific inhibitor of glycolysis, triggered caspase-dependent apoptosis, reduced lactate generation, and blocked the expression of drug resistance-related proteins under low-glucose conditions. These results suggest that the level of ELAVL2/4 is responsible for the development of chemoresistance through activation of the glycolysis pathway under glucose deprivation conditions.

Histopathologic Analysis of Surgically Resected Lungs of Patients with Non-tuberculous Mycobacterial Lung Disease: a Retrospective and Hypothesis-generating Study.

Non-tuberculous mycobacterial lung disease (NTM-LD) is most commonly due to species within the Mycobacterium avium complex (MAC) and Mycobacterium abscessus complex (MAbC). Surgical lung resection, typically a lobectomy or segmentectomy, is occasionally undertaken for individuals with recalcitrant but localized NTM-LD. Since the growth characteristics of MAC (slow growers) and MAbC (rapid growers) as well as their drug susceptibility patterns are significantly different, the objective of this study is to characterize and compare the histopathologic features of the resected lungs due to these two major NTM groups. From 1996 to 2017, 356 patients with NTM-LD due to MAC (n=270), MAbC (n=54), or both (n=32) underwent a total of 404 lobar resections (with the lingula counted as a separate lobe) at the University of Colorado Hospital. We analyzed by microscopy the existing surgical lung tissue sections for bronchiolitis, bronchiolectasis, bronchiectasis, non-necrotizing granuloma (airway, parenchymal, and total), necrotizing granuloma (airway, parenchymal, and total), peri-airway fibrosis, fibrous pleuritis, and lymphoid follicles. There were no significant differences in the presence or absence of most of the histopathologic features of surgically removed lungs due to MAC, MAbC, or both MAC + MAbC. However, there were significantly more necrotizing granulomas (airway, parenchymal, and total) and fibrous pleuritis in MAC compared to MAbC lung diseases. Since necrotizing granulomas may be a sign of inadequate control of the infection, we posit that their presence may be an indication of increased chronicity, increased virulence of MAC compared to MAbC, and/or impaired host immunity against the NTM. Futures studies to determine the root cause of such differences in histopathologic findings in MAC versus MAbC lung disease may spawn new leads on differential pathogenic mechanisms with different NTM, with the goal of aiming for more targeted therapy against both the NTM and the lung damage induced by them.

Peptide transporter2 (PTR2) enhances water uptake during early seed germination in Arabidopsis thaliana.

KEY MESSAGE: PTR2 in Arabidopsis thaliana is negatively regulated by ABI4 and plays a key role in water uptake by seeds, ensuring that imbibed seeds proceed to germination. Peptide transporters (PTRs) transport nitrogen-containing substrates in a proton-dependent manner. Among the six PTRs in Arabidopsis thaliana, the physiological role of the tonoplast-localized, seed embryo abundant PTR2 is unknown. In the present study, a molecular physiological analysis of PTR2 was conducted using ptr2 mutants and PTR2CO complementation lines. Compared with the wild type, the ptr2 mutant showed ca. 6 h delay in testa rupture and consequently endosperm rupture because of 17% lower water content and 10% higher free abscisic acid (ABA) content. Constitutive overexpression of the PTR2 gene under the control of the Cauliflower mosaic virus (CaMV) 35S promoter in ptr2 mutants rescued the mutant phenotypes. After cold stratification, a transient increase in ABA INSENSITIVE4 (ABI4) transcript levels during induction of testa rupture was followed by a similar increase in PTR2 transcript levels, which peaked prior to endosperm rupture. The PTR2 promoter region containing multiple CCAC motifs was recognized by ABI4 in electrophoretic mobility shift assays, and PTR2 expression was repressed by 67% in ABI4 overexpression lines compared with the wild type, suggesting that PTR2 is an immediate downstream target of ABI4. Taken together, the results suggest that ABI4-dependent temporal regulation of PTR2 expression may influence water status during seed germination to promote the post-germinative growth of imbibed seeds.

Structural and functional similarities and differences in nucleolar Pumilio RNA-binding proteins between Arabidopsis and the charophyte Chara corallina.

BACKGROUND: Pumilio RNA-binding proteins are evolutionarily conserved throughout eukaryotes and are involved in RNA decay, transport, and translation repression in the cytoplasm. Although a majority of Pumilio proteins function in the cytoplasm, two nucleolar forms have been reported to have a function in rRNA processing in Arabidopsis. The species of the genus Chara have been known to be most closely related to land plants, as they share several characteristics with modern Embryophyta. RESULTS: In this study, we identified two putative nucleolar Pumilio protein genes, namely, ChPUM2 and ChPUM3, from the transcriptome of Chara corallina. Of the two ChPUM proteins, ChPUM2 was most similar in amino acid sequence (27% identity and 45% homology) and predicted protein structure to Arabidopsis APUM23, while ChPUM3 was similar to APUM24 (35% identity and 54% homology). The transient expression of 35S:ChPUM2-RFP and 35S:ChPUM3-RFP showed nucleolar localization of fusion proteins in tobacco leaf cells, similar to the expression of 35S:APUM23-GFP and 35S:APUM24-GFP. Moreover, 35S:ChPUM2 complemented the morphological defects of the apum23 phenotypes but not those of apum24, while 35S:ChPUM3 could not complement the apum23 and apum24 mutants. Similarly, the 35S:ChPUM2/apum23 plants rescued the pre-rRNA processing defect of apum23, but 35S:ChPUM3/apum24+/- plants did not rescue that of apum24. Consistent with these complementation results, a known target RNA-binding sequence at the end of the 18S rRNA (5'-GGAAUUGACGG) for APUM23 was conserved in Arabidopsis and C. corallina, whereas a target region of ITS2 pre-rRNA for APUM24 was 156 nt longer in C. corallina than in A. thaliana. Moreover, ChPUM2 and APUM23 were predicted to have nearly identical structures, but ChPUM3 and APUM24 have different structures in the 5th C-terminal Puf RNA-binding domain, which had a longer random coil in ChPUM3 than in APUM24. CONCLUSIONS: ChPUM2 of C. corallina was functional in Arabidopsis, similar to APUM23, but ChPUM3 did not substitute for APUM24 in Arabidopsis. Protein homology modeling showed high coverage between APUM23 and ChPUM2, but displayed structural differences between APUM24 and ChPUM3. Together with the protein structure of ChPUM3 itself, a short ITS2 of Arabidopsis pre-rRNA may interrupt the binding of ChPUM3 to 3'-extended 5.8S pre-rRNA.

Qualitative Analysis of the Disease Experience of Korean Older Men With Chronic Obstructive Pulmonary Disease.

The current qualitative study explored perceptions and experiences of chronic obstructive pulmonary disease (COPD) among Korean male older adults. Six older adults participated in a narrative group interview. Responses were analyzed to describe perceptions and experiences of COPD. Social stigma, blaming others and the environment, stress to bear alone, and adaptation and management emerged as relevant themes for adaptation to COPD. In relation to obstacles to healthy behaviors after adapting to COPD, emergent themes were adapting to symptoms, external environmental factors, alternative treatments, and insufficient resources. Facilitators of healthy behaviors were past symptom experience, personal volition, and advice from health professionals. Older adult men with COPD trying to adapt to the disease need sufficient resources and social support from families, social networks, and health professionals. Development of interventions for older adults with COPD should include knowledge and understanding of experience and needs. [Journal of Gerontological Nursing, 46(2), 49-56.].

An Arabidopsis divergent pumilio protein, APUM24, is essential for embryogenesis and required for faithful pre-rRNA processing.

Pumilio RNA-binding proteins are largely involved in mRNA degradation and translation repression. However, a few evolutionarily divergent Pumilios are also responsible for proper pre-rRNA processing in human and yeast. Here, we describe an essential Arabidopsis nucleolar Pumilio, APUM24, that is expressed in tissues undergoing rapid proliferation and cell division. A T-DNA insertion for APUM24 did not affect the male and female gametogenesis, but instead resulted in a negative female gametophytic effect on zygotic cell division immediately after fertilization. Additionally, the mutant embryos displayed defects in cell patterning from pro-embryo through globular stages. The mutant embryos were marked by altered auxin maxima, which were substantiated by the mislocalization of PIN1 and PIN7 transporters in the defective embryos. Homozygous apum24 callus accumulates rRNA processing intermediates, including uridylated and adenylated 5.8S and 25S rRNA precursors. An RNA-protein interaction assay showed that the histidine-tagged recombinant APUM24 binds RNAin vitro with no apparent specificity. Overall, our results demonstrated that APUM24 is required for rRNA processing and early embryogenesis in Arabidopsis.

Genomic Survey and Biochemical Analysis of Recombinant Candidate Cyanobacteriochromes Reveals Enrichment for Near UV/Violet Sensors in the Halotolerant and Alkaliphilic Cyanobacterium Microcoleus IPPAS B353.

Cyanobacteriochromes (CBCRs), which are exclusive to and widespread among cyanobacteria, are photoproteins that sense the entire range of near-UV and visible light. CBCRs are related to the red/far-red phytochromes that utilize linear tetrapyrrole (bilin) chromophores. Best characterized from the unicellular cyanobacterium Synechocystis sp. PCC 6803 and the multicellular heterocyst forming filamentous cyanobacteria Nostoc punctiforme ATCC 29133 and Anabaena sp. PCC 7120, CBCRs have been poorly investigated in mat-forming, nonheterocystous cyanobacteria. In this study, we sequenced the genome of one of such species, Microcoleus IPPAS B353 (Microcoleus B353), and identified two phytochromes and seven CBCRs with one or more bilin-binding cGMP-specific phosphodiesterase, adenylyl cyclase and FhlA (GAF) domains. Biochemical and spectroscopic measurements of 23 purified GAF proteins from phycocyanobilin (PCB) producing recombinant Escherichia coli indicated that 13 of these proteins formed near-UV and visible light-absorbing covalent adducts: 10 GAFs contained PCB chromophores, whereas three contained the PCB isomer, phycoviolobilin (PVB). Furthermore, the complement of Microcoleus B353 CBCRs is enriched in near-UV and violet sensors, but lacks red/green and green/red CBCRs that are widely distributed in other cyanobacteria. We hypothesize that enrichment in short wavelength-absorbing CBCRs is critical for acclimation to high-light environments where this organism is found.

A wheat R2R3-MYB protein PURPLE PLANT1 (TaPL1) functions as a positive regulator of anthocyanin biosynthesis.

Transcriptional activation of anthocyanin biosynthesis genes in vegetative tissues of monocotyledonous plants is mediated by cooperative activity of one component from each of the following two transcription factor families: MYB encoded by PURPLE PLANT1/COLORED ALEURONE1 (PL1/C1), and basic helix-loop-helix (bHLH) encoded by RED/BOOSTER (R1/B1). In the present study, putative PL cDNA was cloned from the wheat (Triticum aestivum) cultivar Iksan370, which preferentially expresses anthocyanins in coleoptiles. Phylogenetic tree analysis of deduced amino acid sequences showed that a putative TaPL1 is highly homologous to barley (Hordeum vulgare) HvPL1, but is distinct from wheat TaC1. Transgenic Arabidopsis thaliana stably expressing putative TaPL1 accumulated anthocyanin pigments in leaves and up-regulated structural genes involved in both early and late anthocyanin biosynthesis steps. TaPL1 transcript levels in Iksan370 were more prominent in vegetative tissues such as young coleoptiles than in reproductive tissues such as spikelets. TaPL1 expression was significantly up-regulated by environmental stresses including cold, salt, and light, which are known to induce anthocyanin accumulation. These combined results suggest that TaPL1 is an active positive regulator of anthocyanin biosynthesis in wheat coleoptiles.

Identification of candidate domestication regions in the radish genome based on high-depth resequencing analysis of 17 genotypes.

KEY MESSAGE: This study provides high-quality variation data of diverse radish genotypes. Genome-wide SNP comparison along with RNA-seq analysis identified candidate genes related to domestication that have potential as trait-related markers for genetics and breeding of radish. Radish (Raphanus sativus L.) is an annual root vegetable crop that also encompasses diverse wild species. Radish has a long history of domestication, but the origins and selective sweep of cultivated radishes remain controversial. Here, we present comprehensive whole-genome resequencing analysis of radish to explore genomic variation between the radish genotypes and to identify genetic bottlenecks due to domestication in Asian cultivars. High-depth resequencing and multi-sample genotyping analysis of ten cultivated and seven wild accessions obtained 4.0 million high-quality homozygous single-nucleotide polymorphisms (SNPs)/insertions or deletions. Variation analysis revealed that Asian cultivated radish types are closely related to wild Asian accessions, but are distinct from European/American cultivated radishes, supporting the notion that Asian cultivars were domesticated from wild Asian genotypes. SNP comparison between Asian genotypes identified 153 candidate domestication regions (CDRs) containing 512 genes. Network analysis of the genes in CDRs functioning in plant signaling pathways and biochemical processes identified group of genes related to root architecture, cell wall, sugar metabolism, and glucosinolate biosynthesis. Expression profiling of the genes during root development suggested that domestication-related selective advantages included a main taproot with few branched lateral roots, reduced cell wall rigidity and favorable taste. Overall, this study provides evolutionary insights into domestication-related genetic selection in radish as well as identification of gene candidates with the potential to act as trait-related markers for background selection of elite lines in molecular breeding.

Elucidating the triplicated ancestral genome structure of radish based on chromosome-level comparison with the Brassica genomes.

KEYMESSAGE: This study presents a chromosome-scale draft genome sequence of radish that is assembled into nine chromosomal pseudomolecules. A comprehensive comparative genome analysis with the Brassica genomes provides genomic evidences on the evolution of the mesohexaploid radish genome. Radish (Raphanus sativus L.) is an agronomically important root vegetable crop and its origin and phylogenetic position in the tribe Brassiceae is controversial. Here we present a comprehensive analysis of the radish genome based on the chromosome sequences of R. sativus cv. WK10039. The radish genome was sequenced and assembled into 426.2 Mb spanning >98 % of the gene space, of which 344.0 Mb were integrated into nine chromosome pseudomolecules. Approximately 36 % of the genome was repetitive sequences and 46,514 protein-coding genes were predicted and annotated. Comparative mapping of the tPCK-like ancestral genome revealed that the radish genome has intermediate characteristics between the Brassica A/C and B genomes in the triplicated segments, suggesting an internal origin from the genus Brassica. The evolutionary characteristics shared between radish and other Brassica species provided genomic evidences that the current form of nine chromosomes in radish was rearranged from the chromosomes of hexaploid progenitor. Overall, this study provides a chromosome-scale draft genome sequence of radish as well as novel insight into evolution of the mesohexaploid genomes in the tribe Brassiceae.

Identification of genes that may regulate the expression of the transcription factor production of anthocyanin pigment 1 (PAP1)/MYB75 involved in Arabidopsis anthocyanin biosynthesis.

KEY MESSAGE: A putative RNA-binding protein with a single RNA Recognition Motif (At3G63450) is involved in anthocyanin biosynthesis via its ability to modulate the transcript level of a major positive regulator PAP1 in Arabidopsis. The R2R3 MYB-activator production of anthocyanin pigment 1 (PAP1)/MYB75 plays a major role in anthocyanin biosynthesis in Arabidopsis in combination with one of three bHLH activators including transparent test 8 (TT8), enhancer of glabra3 (EGL3), glabra3 (GL3), and the WD-repeat transcription factor transparent testa 1 (TTG1), forming ternary MYB-basic HLH-WD40 complexes. Transcriptional activation of PAP1 expression is largely triggered by changes in light color and intensity, temperature fluctuations, nutrient status, and sugar and hormone treatments. However, the immediate upstream and downstream regulatory factors for PAP1 transcription are largely unknown. In the present study, using a T-DNA insertional mutagenesis approach, we transformed pap1-Dominant (pap1D) plants to modulate the levels of endogenous PAP1 transcripts. We employed Restriction Site Extension (RSE)-PCR analysis of 247 homogenous T3 genetic mutant lines exhibiting variations in anthocyanin accumulation compared to pap1D and identified 92 lines with T-DNA integrated in either intra- or inter-genic locations. This analysis revealed 80 novel candidate proteins, including a putative RNA-binding protein with a single RNA Recognition Motif (At3G63450), which may directly or indirectly regulate PAP1 expression at the transcriptional level.

OsMYB14, an R2R3-MYB transcription factor, regulates plant height through the control of hormone metabolism in rice.

Plant growth must be regulated throughout the plant life cycle. The myeloblastosis (MYB) transcription factor (TF) family is one of the largest TF families and is involved in metabolism, lignin biosynthesis, and developmental processes. Here, we showed that OsMYB14, a rice R2R3-MYB TF, was expressed in leaves and roots, especially in rice culm and panicles, and that it localized to the nucleus. Overexpression of OsMYB14 (OsMYB14-ox) in rice resulted in a 30% reduction in plant height compared to that of the wild type (WT), while the height of the osmyb14-knockout (osmyb14-ko) mutant generated using the CRISPR/Cas9 system was not significantly different. Microscopic observations of the first internode revealed that the cell size did not differ significantly among the lines. RNA sequencing analysis revealed that genes associated with plant development, regulation, lipid metabolism, carbohydrate metabolism, and gibberellin (GA) and auxin metabolic processes were downregulated in the OsMYB14-ox line. Hormone quantitation revealed that inactive GA19 accumulated in OsMYB14-ox but not in the WT or knockout plants, suggesting that GA20 generation was repressed. Indole-3-acetic acid (IAA) and IAA-aspartate accumulated in OsMYB14-ox and osmyb14-ko, respectively. Indeed, real-time PCR analysis revealed that the expression of OsGA20ox1, encoding GA20 oxidase 1, and OsGH3-2, encoding IAA-amido synthetase, was downregulated in OsMYB14-ox and upregulated in osmyb14-ko. A protein-binding microarray revealed the presence of a consensus DNA-binding sequence, the ACCTACC-like motif, in the promoters of the OsGA20ox1 and GA20ox2 genes. These results suggest that OsMYB14 may act as a negative regulator of biological processes affecting plant height in rice by regulating GA biosynthesis and auxin metabolism.

Genome-wide binding-site analysis of REVOLUTA reveals a link between leaf patterning and light-mediated growth responses.

Unlike the situation in animals, the final morphology of the plant body is highly modulated by the environment. During Arabidopsis development, intrinsic factors provide the framework for basic patterning processes. CLASS III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIPIII) transcription factors are involved in embryo, shoot and root patterning. During vegetative growth HD-ZIPIII proteins control several polarity set-up processes such as in leaves and the vascular system. We have identified several direct target genes of the HD-ZIPIII transcription factor REVOLUTA (REV) using a chromatin immunoprecipitation/DNA sequencing (ChIP-Seq) approach. This analysis revealed that REV acts upstream of auxin biosynthesis and affects directly the expression of several class II HD-ZIP transcription factors that have been shown to act in the shade-avoidance response pathway. We show that, as well as involvement in basic patterning, HD-ZIPIII transcription factors have a critical role in the control of the elongation growth that is induced when plants experience shade. Leaf polarity is established by the opposed actions of HD-ZIPIII and KANADI transcription factors. Finally, our study reveals that the module that consists of HD-ZIPIII/KANADI transcription factors controls shade growth antagonistically and that this antagonism is manifested in the opposed regulation of shared target genes.

Calcium dependent sucrose uptake links sugar signaling to anthocyanin biosynthesis in Arabidopsis.

Sugars enhance light signaling-induced anthocyanin accumulation in Arabidopsis seedlings via differential regulation of several positive and negative transcription factors. Ca(2+) plays a role as a second messenger in sugar signaling in grape and wheat. However, whether anthocyanin pigmentation is modulated by changes in intracellular Ca(2+) level in Arabidopsis is not known. Here, we used a pharmaceutical approach that Ca(2+) antagonists strongly interfered with sucrose uptake and anthocyanin accumulation by downregulating the expression of sucrose transporter 1 (SUC1) and transcriptional regulatory factors, such as PAP1. Time course analysis of the effect of Ca(2+) antagonists showed the early inhibition of sucrose-induced sugar uptake leading to decreased anthocyanin accumulation, indicating that Ca(2+) signals play a role in sugar uptake rather than in anthocyanin biosynthesis. An early increase in cytosolic Ca(2+) level in Arabidopsis roots in response to sucrose feeding was significantly inhibited by Ca(2+) antagonists. Taken together, these results indicate that sucrose-induced sugar uptake in Arabidopsis is modulated by changes in endogenous Ca(2+) levels, which in turn regulate anthocyanin accumulation.

Bruno-like proteins modulate flowering time via 3' UTR-dependent decay of SOC1 mRNA.

The Bruno RNA-binding protein (RBP) has been shown to initially repress the translation of oskar mRNA during Drosophila oogenesis and later to be involved in a broad range of RNA regulation. Here, we show that homologous constitutive overexpression of each of two Arabidopsis thaliana Bruno-like genes, AtBRN1 and AtBRN2, delayed the flowering time, while the atbrn1 atbrn2-3 double mutant flowered early and exhibited increased expression of APETALA1 (AP1) and LEAFY (LFY) transcripts. Crossing of 35S::AtBRNs with SOC1 101-D plants demonstrated that 35S::AtBRNs suppress an early-flowering phenotype of SOC1 101-D in which the coding sequence (CDS) with the 3' UTR of SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1 (SOC1) gene is overexpressed. However, this early-flowering phenotype by SOC1 overexpression was maintained in the plants coexpressing 35S::AtBRNs and 35S::SOC1 without the 3' UTR (-3' UTR). Using yeast three-hybrid, electrophoretic mobility shift, RNA immunoprecipitation, and protoplast transient assays, we found that AtBRNs bind to the 3' UTR of SOC1 RNA and participate in mRNA decay, which was mediated by the distal region of the SOC1 3' UTR. Overall, AtBRNs repress SOC1 activity in a 3' UTR-dependent manner, thereby controlling the flowering time in Arabidopsis.

The Phytophthora nucleolar effector Pi23226 targets host ribosome biogenesis to induce necrotrophic cell death.

Pathogen effectors target diverse subcellular organelles to manipulate the plant immune system. Although the nucleolus has emerged as a stress marker and several effectors are localized in the nucleolus, the roles of nucleolar-targeted effectors remain elusive. In this study, we showed that Phytophthora infestans infection of Nicotiana benthamiana results in nucleolar inflation during the transition from the biotrophic to the necrotrophic phase. Multiple P. infestans effectors were localized in the nucleolus: Pi23226 induced cell death in N. benthamiana and nucleolar inflation similar to that observed in the necrotrophic stage of infection, whereas its homolog Pi23015 and a deletion mutant (Pi23226ΔC) did not induce cell death or affect nucleolar size. RNA immunoprecipitation and individual-nucleotide-resolution UV crosslinking and immunoprecipitation sequencing analysis indicated that Pi23226 bound to the 3' end of 25S rRNA precursors, resulting in accumulation of unprocessed 27S pre-rRNAs. The nucleolar stress marker NAC082 was strongly upregulated under Pi23226-expressing conditions. Pi23226 subsequently inhibited global protein translation in host cells by interacting with ribosomes. Pi23226 enhanced P. infestans pathogenicity, indicating that Pi23226-induced ribosome malfunction and cell death were beneficial for pathogenesis in the host. Our results provide evidence for the molecular mechanism underlying RNA-binding effector activity in host ribosome biogenesis and lead to new insights into the nucleolar action of effectors in pathogenesis.

Impaired function of the tonoplast-localized sucrose transporter in rice, OsSUT2, limits the transport of vacuolar reserve sucrose and affects plant growth.

Physiological functions of sucrose (Suc) transporters (SUTs) localized to the tonoplast in higher plants are poorly understood. We here report the isolation and characterization of a mutation in the rice (Oryza sativa) OsSUT2 gene. Expression of OsSUT2-green fluorescent protein in rice revealed that OsSUT2 localizes to the tonoplast. Analysis of the OsSUT2 promoter::ß-glucuronidase transgenic rice indicated that this gene is highly expressed in leaf mesophyll cells, emerging lateral roots, pedicels of fertilized spikelets, and cross cell layers of seed coats. Results of Suc transport assays in yeast were consistent with a H(+)-Suc symport mechanism, suggesting that OsSUT2 functions in Suc uptake from the vacuole. The ossut2 mutant exhibited a growth retardation phenotype with a significant reduction in tiller number, plant height, 1,000-grain weight, and root dry weight compared with the controls, the wild type, and complemented transgenic lines. Analysis of primary carbon metabolites revealed that ossut2 accumulated more Suc, glucose, and fructose in the leaves than the controls. Further sugar export analysis of detached leaves indicated that ossut2 had a significantly decreased sugar export ability compared with the controls. These results suggest that OsSUT2 is involved in Suc transport across the tonoplast from the vacuole lumen to the cytosol in rice, playing an essential role in sugar export from the source leaves to sink organs.

APUM23, a nucleolar Puf domain protein, is involved in pre-ribosomal RNA processing and normal growth patterning in Arabidopsis.

Pumilio, an RNA-binding protein that contains tandemly repeated Puf domains, is known to repress translational activity in early embryogenesis and polarized cells of non-plant species. Although Pumilio proteins have been characterized in many eukaryotes, their role in plants is unknown. In the present study, we characterized an Arabidopsis Pumilio-encoding gene, APUM23. APUM23 is constitutively expressed, with higher levels in metabolically active tissues, and its expression is up-regulated in the presence of either glucose or sucrose. The T-DNA insertion mutants apum23-1 and apum23-2 showed slow growth, with serrated and scrunched leaves, an abnormal venation pattern, and distorted organization of the palisade parenchyma cells - a phenotype that is reminiscent of nucleolin and ribosomal protein gene mutants. Intracellular localization studies indicate that APUM23 predominantly localizes to the nucleolus. Based on this localization, rRNA processing was examined. In apum23, 35S pre-rRNA, and unprocessed 18S and 5.8S poly(A) rRNAs, accumulated without affecting the steady-state levels of mature rRNAs, indicating that APUM23 is involved in the processing and/or degradation of 35S pre-rRNA and rRNA maturation by-products. The apum23 mutant showed increased levels of 18S rRNA biogenesis-related U3 and U14 small nucleolar RNAs (snoRNAs) and accumulated RNAs within the nucleolus. Our data suggest that APUM23 plays an important role in plant development via rRNA processing.

The DOF transcription factor Dof5.1 influences leaf axial patterning by promoting Revoluta transcription in Arabidopsis.

Dof proteins are transcription factors that have a conserved single zinc finger DNA-binding domain. In this study, we isolated an activation tagging mutant Dof5.1-D exhibiting an upward-curling leaf phenotype due to enhanced expression of the REV gene that is required for establishing adaxial-abaxial polarity. Dof5.1-D plants also had reduced transcript levels for IAA6 and IAA19 genes, indicating an altered auxin biosynthesis in Dof5.1-D. An electrophoretic mobility shift assay using the Dof5.1 DNA-binding motif and the REV promoter region indicated that the DNA-binding domain of Dof5.1 binds to a TAAAGT motif located in the 5'-distal promoter region of the REV promoter. Further, transient and chromatin immunoprecipitation assays verified binding activity of the Dof5.1 DNA-binding motif with the REV promoter. Consistent with binding assays, constitutive over-expression of the Dof5.1 DNA-binding domain in wild-type plants caused a downward-curling phenotype, whereas crossing Dof5.1-D to a rev mutant reverted the upward-curling phenotype of the Dof5.1-D mutant leaf to the wild-type. These results suggest that the Dof5.1 protein directly binds to the REV promoter and thereby regulates adaxial-abaxial polarity.