Nsp14 of SARS-CoV-2 inhibits mRNA processing and nuclear export by targeting the nuclear cap-binding complex.

To facilitate selfish replication, viruses halt host gene expression in various ways. The nuclear export of mRNA is one such process targeted by many viruses. SARS-CoV-2, the etiological agent of severe acute respiratory syndrome, also prevents mRNA nuclear export. In this study, Nsp14, a bifunctional viral replicase subunit, was identified as a novel inhibitor of mRNA nuclear export. Nsp14 induces poly(A)+ RNA nuclear accumulation and the dissolution/coalescence of nuclear speckles. Genome-wide gene expression analysis revealed the global dysregulation of splicing and 3'-end processing defects of replication-dependent histone mRNAs by Nsp14. These abnormalities were also observed in SARS-CoV-2-infected cells. A mutation introduced at the guanine-N7-methyltransferase active site of Nsp14 diminished these inhibitory activities. Targeted capillary electrophoresis-mass spectrometry analysis (CE-MS) unveiled the production of N7-methyl-GTP in Nsp14-expressing cells. Association of the nuclear cap-binding complex (NCBC) with the mRNA cap and subsequent recruitment of U1 snRNP and the stem-loop binding protein (SLBP) were impaired by Nsp14. These data suggest that the defects in mRNA processing and export arise from the compromise of NCBC function by N7-methyl-GTP, thus exemplifying a novel viral strategy to block host gene expression.

A simple method for creating transgenic pea hairy roots using a Japanese pea cultivar and a Japanese Rhizobium radiobacter strain.

Pea (Pisum sativum) is an agriculturally important leguminous crop cultivated worldwide. It is also the plant from which phytoalexin was isolated for the first time. Several studies have investigated gene functions using pea hairy root culture systems. However, the procedures for producing hairy roots are relatively complicated and only a few pea cultivars and Rhizobium strains have been used. In this study, we established a simple method for generating transgenic hairy roots using a pea cultivar and a Rhizobium strain available in Japan. The transformation efficiency for the transgenic hairy roots (approximately 14%) was calculated on the basis of GFP fluorescence because the binary vector used in this study carried a GFP cassette as a marker. Furthermore, we confirmed that the production of the phytoalexin (+)-pisatin was induced by a copper dichloride treatment, indicating that this system can be used to characterize the biosynthesis of (+)-pisatin, which is a compound with a unique pterocarpan structure. Interestingly, some of the hairy roots turned into crown galls during the culture period. In summary, our simple method enables the production of transgenic pea hairy roots using biological materials accessible in Japan. The generated hairy roots can be used to elucidate the molecular mechanisms underlying (+)-pisatin biosynthesis as well as hairy root/crown gall formation.

Nicotinamide mononucleotide and related metabolites induce disease resistance against fungal phytopathogens in Arabidopsis and barley.

Nicotinamide mononucleotide (NMN), a precursor of nicotinamide adenine dinucleotide (NAD), is known to act as a functional molecule in animals, whereas its function in plants is largely unknown. In this study, we found that NMN accumulated in barley cultivars resistant to phytopathogenic fungal Fusarium species. Although NMN does not possess antifungal activity, pretreatment with NMN and related metabolites enhanced disease resistance to Fusarium graminearum in Arabidopsis leaves and flowers and in barley spikes. The NMN-induced Fusarium resistance was accompanied by activation of the salicylic acid-mediated signalling pathway and repression of the jasmonic acid/ethylene-dependent signalling pathways in Arabidopsis. Since NMN-induced disease resistance was also observed in the SA-deficient sid2 mutant, an SA-independent signalling pathway also regulated the enhanced resistance induced by NMN. Compared with NMN, NAD and NADP, nicotinamide pretreatment had minor effects on resistance to F. graminearum. Constitutive expression of the NMNAT gene, which encodes a rate-limiting enzyme for NAD biosynthesis, resulted in enhanced disease resistance in Arabidopsis. Thus, modifying the content of NAD-related metabolites can be used to optimize the defence signalling pathways activated in response to F. graminearum and facilitates the control of disease injury and mycotoxin accumulation in plants.

Co-ordinated Regulations of mRNA Synthesis and Decay during Cold Acclimation in Arabidopsis Cells.

Plants possess a cold acclimation system to acquire freezing tolerance through pre-exposure to non-freezing low temperatures. The transcriptional cascade of C-repeat-binding factors (CBFs)/dehydration response element-binding factors (DREBs) is considered a major transcriptional regulatory pathway during cold acclimation. However, little is known regarding the functional significance of mRNA stability regulation in the response of gene expression to cold stress. The actual level of individual mRNAs is determined by a balance between mRNA synthesis and degradation. Therefore, it is important to assess the regulatory steps to increase our understanding of gene regulation. Here, we analyzed temporal changes in mRNA amounts and half-lives in response to cold stress in Arabidopsis cell cultures based on genome-wide analysis. In this mRNA decay array method, mRNA half-life measurements and microarray analyses were combined. In addition, temporal changes in the integrated value of transcription rates were estimated from the above two parameters using a mathematical approach. Our results showed that several cold-responsive genes, including Cold-regulated 15a, were relatively destabilized, whereas the mRNA amounts were increased during cold treatment by accelerating the transcription rate to overcome the destabilization. Considering the kinetics of mRNA synthesis and degradation, this apparently contradictory result supports that mRNA destabilization is advantageous for the swift increase in CBF-responsive genes in response to cold stress.

ACR11 is an Activator of Plastid-Type Glutamine Synthetase GS2 in Arabidopsis thaliana.

Glutamine synthetase (GS) is an important enzyme for nitrogen assimilation, and GS2, encoded by GLN2, is the only plastid-type GS in Arabidopsis thaliana. A co-expression analysis suggested that the expression level of the gene encoding a uridylyltransferase-like protein, ACR11, is strongly correlated with GLN2 expression levels. Here we showed that the recombinant ACR11 protein increased GS2 activity in vitro by reducing the Km values of its substrate glutamine. A T-DNA insertion mutant of ACR11 exhibited a reduced GS activity under low nitrate conditions and reduced glutamine levels. Biochemical analyses revealed that ACR11 and GS2 interacted both in vitro and in vivo. These data demonstrate that ACR11 is an activator of GS2, giving it a mechanistic role in the nitrogen assimilation of A. thaliana.

WRKY6 is involved in the response to boron deficiency in Arabidopsis thaliana.

Boron (B) is one of the essential nutrients for plant growth and reproduction. Transcriptome analyses have identified genes regulated by B deficiency, but their function mostly remains elusive. To identify the functions of B deficiency-inducible genes, T-DNA insertion mutants of 10 B deficiency-induced genes were obtained, and their growth properties in response to B conditions were examined. Among the lines examined, mutants of the transcription factor WRKY6 showed growth defect compared with the wild-type under B deficiency, but not under normal conditions. This growth defect was commonly observed among three independently isolated wrky6 mutants. There was no significant difference in B concentration between wrky6-3 and the wild-type. Promoter activity of WRKY6 was induced around the root tip under B deficiency. These results established that WRKY6 is a low-B-induced transcription factor gene that is essential for normal root growth under low-B conditions. Transcriptome analysis around the root tip identified WRKY6-regulated genes under B deficiency. Our findings represent the first identification of a transcription factor involved in the response to B deficiency.