BAH1/NLA, a RING-Type Ubiquitin E3 Ligase, Regulates the Accumulation of Salicylic Acid and Immune Responses to Pseudomonas syringae DC30001[W][OA]

Salicylic acid (SA) is a primary factor responsible for exerting diverse immune responses in plants and is synthesized in response to attack by a wide range of pathogens. The Arabidopsis (Arabidopsis thaliana) sid2 mutant is defective in a SA biosynthetic pathway involving ISOCHORISMATE SYNTHASE1 (ICS1) and consequently contains reduced levels of SA. However, the sid2 mutant as well as ICS-suppressed tobacco (Nicotiana benthamiana) still accumulate a small but significant level of SA. These observations along with previous studies suggest that SA might also be synthesized by another pathway involving benzoic acid (BA). Here we isolated a benzoic acid hypersensitive1-Dominant (bah1-D) mutant that excessively accumulated SA after application of BA from activation-tagged lines. This mutant also accumulated higher levels of SA after inoculation with Pseudomonas syringae pv tomato DC3000. Analysis of the bah1-D sid2 double mutant suggested that the bah1-D mutation caused both ICS1-dependent and -independent accumulation. In addition, the bah1-D mutant showed SA-dependent localized cell death in response to P. syringae pv tomato DC3000. The T-DNA insertional mutation that caused the bah1-D phenotypes resulted in the suppression of expression of the NLA gene, which encodes a RING-type ubiquitin E3 ligase. These results suggest that BAH1/NLA plays crucial roles in the ubiquitination-mediated regulation of immune responses, including BA- and pathogen-induced SA accumulation, and control of cell death

Knockout of Tobacco Homologs of <i>Arabidopsis Multi-Antibiotic Resistance 1</i> Gene Confers a Limited Resistance to Aminoglycoside Antibiotics

To explore a possible recessive selective marker for future DNA-free genome editing by direct delivery of a CRISPR/Cas9-single guide RNA (sgRNA) ribonucleoprotein complex, we knocked out homologs of the ArabidopsisMulti-Antibiotic Resistance 1 (MAR1)/RTS3 gene, mutations of which confer aminoglycoside resistance, in tobacco plants by an efficient Agrobacterium-mediated gene transfer. A Cas9 gene was introduced into Nicotiana tabacum and Nicotiana sylvestris together with an sgRNA gene for one of three different target sequences designed to perfectly match sequences in both S- and T-genome copies of N. tabacumMAR1 homologs (NtMAR1hs). All three sgRNAs directed the introduction of InDels into NtMAR1hs, as demonstrated by CAPS and amplicon sequencing analyses, albeit with varying efficiency. Leaves of regenerated transformant shoots were evaluated for aminoglycoside resistance on shoot-induction media containing different aminoglycoside antibiotics. All transformants tested were as sensitive to those antibiotics as non-transformed control plants, regardless of the mutation rates in NtMAR1hs. The NtMAR1hs–knockout seedlings of the T1 generation showed limited aminoglycoside resistance but failed to form shoots when cultured on shoot-induction media containing kanamycin. The results suggest that, like Arabidopsis MAR1, NtMAR1hs have a role in plants’ sensitivity to aminoglycoside antibiotics, and that tobacco has some additional functional homologs

Nicotinamide Effectively Suppresses Fusarium Head Blight in Wheat Plants

Pyridine nucleotides such as a nicotinamide adenine dinucleotide (NAD) are known as plant defense activators. We previously reported that nicotinamide mononucleotide (NMN) enhanced disease resistance against fungal pathogen Fusarium graminearum in barley and Arabidopsis. In this study, we reveal that the pretreatment of nicotinamide (NIM), which does not contain nucleotides, effectively suppresses disease development of Fusarium Head Blight (FHB) in wheat plants. Correspondingly, deoxynivalenol (DON) mycotoxin accumulation was also significantly decreased by NIM pretreatment. A metabolome analysis showed that several antioxidant and antifungal compounds such as trigonelline were significantly accumulated in the NIM-pretreated spikes after inoculation of F. graminearum. In addition, some metabolites involved in the DNA hypomethylation were accumulated in the NIM-pretreated spikes. On the other hand, fungal metabolites DON and ergosterol peroxide were significantly reduced by the NIM pretreatment. Since NIM is relative stable and inexpensive compared with NMN and NAD, it may be more useful for the control of symptoms of FHB and DON accumulation in wheat and other crops

Nicotinamide Mononucleotide Potentiates Resistance to Biotrophic Invasion of Fungal Pathogens in Barley

Nicotinamide mononucleotide (NMN), a precursor of nicotinamide adenine dinucleotide (NAD), induces disease resistance to the Fusarium head blight fungus Fusarium graminearum in Arabidopsis and barley, but it is unknown at which stage of the infection it acts. Since the rate of haustorial formation of an obligate biotrophic barley powdery mildew fungus Blumeria graminis f. sp. hordei (Bgh) was significantly reduced in NMN-treated coleoptile epidermal cells, the possibility that NMN induces resistance to the biotrophic stage of F. graminearum was investigated. The results show that NMN treatment caused the wandering of hyphal growth and suppressed the formation of appressoria-like structures. Furthermore, we developed an experimental system to monitor the early stage of infection in real-time and analyzed the infection behavior. We observed that the hyphae elongated windingly by NMN treatment. These results suggest that NMN potentiates resistance to the biotrophic invasion of F. graminearum as well as Bgh

Fast and Inexpensive Phenotyping and Genotyping Methods for Evaluation of Barley Mutant Population

To further develop barley breeding and genetics, more information on gene functions based on the analysis of the mutants of each gene is needed. However, the mutant resources are not as well developed as the model plants, such as Arabidopsis and rice. Although genome editing techniques have been able to generate mutants, it is not yet an effective method as it can only be used to transform a limited number of cultivars. Here, we developed a mutant population using &lsquo;Mannenboshi&rsquo;, which produces good quality grains with high yields but is susceptible to disease, to establish a Targeting Induced Local Lesions IN Genomes (TILLING) system that can isolate mutants in a high-throughput manner. To evaluate the availability of the prepared 8043 M3 lines, we investigated the frequency of mutant occurrence using a rapid, visually detectable waxy phenotype as an indicator. Four mutants were isolated and single nucleotide polymorphisms (SNPs) were identified in the Waxy gene as novel alleles. It was confirmed that the mutations could be easily detected using the mismatch endonuclease CELI, revealing that a sufficient number of mutants could be rapidly isolated from our TILLING population

Impaired Expression of Chloroplast HSP90C Chaperone Activates Plant Defense Responses with a Possible Link to a Disease-Symptom-Like Phenotype

RNA-seq analysis of a transgenic tobacco plant, i-hpHSP90C, in which chloroplast HSP90C genes can be silenced in an artificially inducible manner resulting in the development of chlorosis, revealed the up- and downregulation of 2746 and 3490 genes, respectively. Gene ontology analysis of these differentially expressed genes indicated the upregulation of ROS-responsive genes; the activation of the innate immunity and cell death pathways; and the downregulation of genes involved in photosynthesis, plastid organization, and cell cycle. Cell death was confirmed by trypan blue staining and electrolyte leakage assay, and the H2O2 production was confirmed by diaminobenzidine staining. The results collectively suggest that the reduced levels of HSP90C chaperone lead the plant to develop chlorosis primarily through the global downregulation of chloroplast- and photosynthesis-related genes and additionally through the light-dependent production of ROS, followed by the activation of immune responses, including cell death

Correction: Islam, S., et al. Impaired Expression of Chloroplast HSP90C Chaperone Activates Plant Defense Responses with a Possible Link to a Disease-Symptom-Like Phenotype. International Journal of Molecular Science 2020, 21, 4202

The authors wish to make the following corrections to this paper [...

Neuroprotective and Anti-Microglial Activation Effects of Tocotrienols in Brains of Lipopolysaccharide-Induced Inflammatory Model Mice

Inflammation is the cause and/or result of many diseases in peripheral tissues and the central nervous system. Recent findings suggested that inflammation in peripheral tissue induces an inflammatory response in the brain that activates glial cells, which, in turn, induce neuronal cell dysfunction. Therefore, anti-inflammatory compounds are important for the suppression of chronic inflammation and prevention of disease. The present study revealed microglial activation in the hippocampus of the brain two days after the peripheral administration of lipopolysaccharide (LPS). Furthermore, the expression of the synaptic vesicle membrane protein, synaptophysin, in the CA3 stratum lucidum of the hippocampus was down-regulated 7 days after the LPS injection. The administration of tocotrienols, a type of vitamin E, significantly attenuated these changes in the hippocampus. Collectively, the present results demonstrated the spread of peripheral inflammatory responses to the brain, in which glial activation and neuronal dysfunction were induced, while tocotrienols exerted anti-inflammatory effects and protected neurons from damage