The transcription factor ORA59 exhibits dual DNA binding specificity that differentially regulates ethylene- and jasmonic acid-induced genes in plant immunity.

Jasmonic acid (JA) and ethylene (ET) signaling modulate plant defense against necrotrophic pathogens in a synergistic and interdependent manner, while JA and ET also have independent roles in certain processes, e.g. in responses to wounding and flooding, respectively. These hormone pathways lead to transcriptional reprogramming, which is a major part of plant immunity and requires the roles of transcription factors. ET response factors are responsible for the transcriptional regulation of JA/ET-responsive defense genes, of which ORA59 functions as a key regulator of this process and has been implicated in the JA-ET crosstalk. We previously demonstrated that Arabidopsis (Arabidopsis thaliana) GDSL LIPASE 1 (GLIP1) depends on ET for gene expression and pathogen resistance. Here, promoter analysis of GLIP1 revealed ERELEE4 as the critical cis-element for ET-responsive GLIP1 expression. In a yeast one-hybrid screening, ORA59 was isolated as a specific transcription factor that binds to the ERELEE4 element, in addition to the well-characterized GCC box. We found that ORA59 regulates JA/ET-responsive genes through direct binding to these elements in gene promoters. Notably, ORA59 exhibited a differential preference for GCC box and ERELEE4, depending on whether ORA59 activation is achieved by JA and ET, respectively. JA and ET induced ORA59 phosphorylation, which was required for both activity and specificity of ORA59. Furthermore, RNA-seq and virus-induced gene silencing analyses led to the identification of ORA59 target genes of distinct functional categories in JA and ET pathways. Our results provide insights into how ORA59 can generate specific patterns of gene expression dynamics through JA and ET hormone pathways.

Identification of the O/ME-SA/Ind-2001e Sublineage of Foot-and-Mouth Disease Virus in Cambodia.

Foot-and-mouth (FMD) is endemic in Cambodia with numerous outbreaks in cattle, pigs and other susceptible animal species reported every year. Historically, these outbreaks were caused by the FMD virus (FMDV) of serotype O PanAsia and Mya-98 lineages and serotype A Sea-97 lineage. However, the trans-pool movement of FMDV between inter-pool regions or countries throughout FMD endemic regions has raised concerns regarding infection with the new genotype or serotype of FMDV in Cambodia. In this study, 19 sequences of VP1 coding region obtained from 33 clinical samples collected from FMDV-affected cattle farms in Cambodia during January to March 2019 were genetically characterized to identify the genotypes/lineages of FMDV. Phylogenetic analysis of VP1 coding sequences revealed that recent field viruses belonged to O/ME-SA/Ind-2001e (15.8%), O/ME-SA/PanAsia (52.7%), and A/ASIA/Sea-97 (31.5%). Besides, the field viruses of O/ME-SA/Ind-2001e in Cambodia showed 93.5-96.8% identity with the VP1 coding sequences of the same sublineage viruses from pool 1 and 2 surrounding Cambodia. This is the first report of O/ME-SA/Ind-2001e infection in Cambodia, suggesting that the trans-pool movement of the new genotype should be closely monitored for efficient control of FMD.

Arabidopsis GDSL lipase 2 plays a role in pathogen defense via negative regulation of auxin signaling.

GLIP1 was isolated previously from Arabidopsis, as a salicylic acid-responsive secreted GDSL lipase that functions in resistance to Alternaria brassicicola [I.S. Oh, A.R. Park, M.S. Bae, S.J. Kwon, Y.S. Kim, J.E. Lee, N.Y. Kang, S. Lee, H. Cheong, O.K. Park, Secretome analysis reveals an Arabidopsis lipase involved in defense against Alternaria brassicicola. Plant Cell 17 (2005) 2832-2847.]. To extend our knowledge of the roles played by GLIPs in Arabidopsis, we conducted functional studies of another family member, GLIP2. GLIP2 transcripts were expressed in young seedlings, as well as in the root and stem tissues of mature plants. GLIP2 transcript levels were elevated by treatment with salicylic acid, jasmonic acid and ethylene. Recombinant GLIP2 proteins possessed lipase and anti-microbial activities, inhibiting germination of fungal spores. In comparison to wild type plants, T-DNA insertion glip2 mutants exhibited enhanced auxin responses, including increased lateral root formation and elevated AUX/IAA gene expression. When challenged with the necrotropic bacteria Erwinia carotovora, glip2 mutants exhibited more susceptible phenotypes than wild type plants. These results suggest that GLIP2 plays a role in resistance to Erwinia carotovora via negative regulation of auxin signaling.

Bioactivity of Fermented Green Coffee Bean Extract Containing High Chlorogenic Acid and Surfactin.

Chlorogenic acid (CGA) is a major component of green coffee beans. Surfactin, a cyclic lipopeptide, is produced and secreted by Bacillus subtilis strains. In this study, bioactivities of fermented green coffee bean extract (FGCBE) and the individual compounds, CGA and surfactin. were compared in HepG2 cells. The concentration of surfactin and CGA in the FGCBE and non-fermented green coffee bean extract (NFGCBE) were determined to be 9.2 and 7.33 and 0.72 and 0.53 mg·mL-1, respectively. The FGCBE contained about 20% and 26% more CGA and surfactin than the NFGCBE. Although CGA and surfactin exhibited cytotoxicity at concentrations more than 100 and 20 µg respectively, the FGCBE 50 containing CGA (460 µg·mL-1) and surfactin (720 µg·mL-1) effectively prevented cell death by oxidative stress and also strongly activated the proliferation of cells incubated with under 50 µM H2O2. The CGA and surfactin in FGCBE were 9.2 and 72 times higher than the CGA and surfactin compounds (50 and 10 µg·mL-1). The relative proliferation of the FGCBE-treated cells also was 3.3 and 8.8 times higher than the CGA and surfactin compounds treated the oxidative stressed cells with 50 µM H2O2. These results suggest that the single compounds such as CGA and surfactin generally have cytotoxicity at low concentration of them but FGCBE contained them acted as strong antioxidants, activators of cell proliferation, inhibitors of cell apoptosis. Various bioactive compounds in fermented coffee bean also seem to help cell proliferation and decreasing of cytotoxicity by CGA and surfactin in coffee bean.

The Arabidopsis Cysteine-Rich Receptor-Like Kinase CRK36 Regulates Immunity through Interaction with the Cytoplasmic Kinase BIK1.

Receptor-like kinases are important signaling components that regulate a variety of cellular processes. In this study, an Arabidopsis cDNA microarray analysis led to the identification of the cysteine-rich receptor-like kinase CRK36 responsive to the necrotrophic fungal pathogen, Alternaria brassicicola. To determine the function of CRK36 in plant immunity, T-DNA-insertion knockdown (crk36) and overexpressing (CRK36OE) plants were prepared. CRK36OE plants exhibited increased hypersensitive cell death and ROS burst in response to avirulent pathogens. Treatment with a typical pathogen-associated molecular pattern, flg22, markedly induced pattern-triggered immune responses, notably stomatal defense, in CRK36OE plants. The immune responses were weakened in crk36 plants. Protein-protein interaction assays revealed the in vivo association of CRK36, FLS2, and BIK1. CRK36 enhanced flg22-triggered BIK1 phosphorylation, which showed defects with Cys mutations in the DUF26 motifs of CRK36. Disruption of BIK1 and RbohD/RbohF genes further impaired CRK36-mediated stomatal defense. We propose that CRK36, together with BIK1 and NADPH oxidases, may form a positive activation loop that enhances ROS burst and leads to the promotion of stomatal immunity.

Cloning, characterization and expression of a Lateral suppressor-like gene from chrysanthemum (Dendranthema grandiflorum Kitamura).

Conventionally, the lateral shoots of chrysanthemum are removed manually, which is time consuming and uneconomical. The development of branchless chrysanthemum will economize its commercial cultivation. To investigate the regulatory mechanism of branchlessness, we undertook cloning of Dendranthema grandiflorum Kitamura Lateral suppressor-like (DgLsL) gene for development of lateral shoot in chrysanthemum. A full-length cDNA of DgLsL gene was isolated by screening cDNA library and performing Rapid Amplification of cDNA Ends (RACE) PCR. Phylogenetic analysis showed that the DgLsL gene is closely related to Lateral suppressor that encodes transcriptional regulator proteins belonging to GRAS family of known transcription factor. Southern blot analysis revealed that DgLsL gene in chrysanthemum genome has one copy. DgLsL expression was apparently up-regulated by ethephon treatment. The expression patterns revealed that DgLsL transcripts were detected in all organs, but showed their highest level in stems.

Differential susceptibility of photosynthesis to light-chilling stress in rice (Oryza sativa l.) depends on the capacity for photochemical dissipation of light.

The primary target for light-chilling stress in chilling-sensitive cucumber leaves is the chloroplast Cu,Zn-Superoxide dismutase, followed by subsequent inactivation of the photosystem (PS) I by reactive oxygen species (ROS). To test this hypothesis, two rice cultivars that were different in their ecological origins (a chilling-resistant Stejaree 45 and a chilling-sensitive Milyang 23) were evaluated with respect to photosynthetic properties, the ROS scavenging system, and expression of genes that are involved in sucrose synthesis and allocation upon the light-chilling stress. As expected, when the leaves were exposed to various low temperatures with illumination (150 micromol m(-2)s(-1)) for 6 h, the leaf photosynthesis of Milyang 23 decreased faster than that of Stejaree 45. The light-chilling induced differential photoinhibition of photosynthesis between the two cultivars was caused by the photoin-activation of PSII but not of PSI, since the potential quantum yield of PSII followed a similar trend to the changes in photosynthetic rates. The activities of the two chloroplastic antioxidant enzymes (superoxide dismutase and ascorbate peroxidase) that are known to be sensitive to oxidative stress were barely affected by the light-chilling treatments. Among various genes in sucrose metabolism (such as cytosolic FBPase, SPS, SUT, SuSy, and AGPase), the transcript levels of SuSy in Milyang 23 were significantly decreased by light-chilling stress compared to that of Stejaree 45. Based on these results, we propose that PSII, not PSI, is the sensitive site for light-chilling stress in chilling-sensitive rice. The extent of PSII photoinhibition depends on its capacity for the photochemical utilization of light.

Disruption of glycosylphosphatidylinositol-anchored lipid transfer protein gene altered cuticular lipid composition, increased plastoglobules, and enhanced susceptibility to infection by the fungal pathogen Alternaria brassicicola.

All aerial parts of vascular plants are covered with cuticular waxes, which are synthesized by extensive export of intracellular lipids from epidermal cells to the surface. Although it has been suggested that plant lipid transfer proteins (LTPs) are involved in cuticular lipid transport, the in planta evidence is still not clear. In this study, a glycosylphosphatidylinositol-anchored LTP (LTPG1) showing higher expression in epidermal peels of stems than in stems was identified from an Arabidopsis (Arabidopsis thaliana) genome-wide microarray analysis. The expression of LTPG1 was observed in various tissues, including the epidermis, stem cortex, vascular bundles, mesophyll cells, root tips, pollen, and early-developing seeds. LTPG1 was found to be localized in the plasma membrane. Disruption of the LTPG1 gene caused alterations of cuticular lipid composition, but no significant changes on total wax and cutin monomer loads were seen. The largest reduction (10 mass %) in the ltpg1 mutant was observed in the C29 alkane, which is the major component of cuticular waxes in the stems and siliques. The reduced content was overcome by increases of the C29 secondary alcohols and C29 ketone wax loads. The ultrastructure analysis of ltpg1 showed a more diffuse cuticular layer structure, protrusions of the cytoplasm into the vacuole in the epidermis, and an increase of plastoglobules in the stem cortex and leaf mesophyll cells. Furthermore, the ltpg1 mutant was more susceptible to infection by the fungus Alternaria brassicicola than the wild type. Taken together, these results indicated that LTPG1 contributed either directly or indirectly to cuticular lipid accumulation.