Inhibitory effect of tannic acid on the growth of Apiospora arundinis and 3-Nitropropionic acid production.

AIMS: This study aimed to investigate the inhibitory effect of tannic acid (TA) on the growth of Apiospora arundinis and 3-Nitropropionic acid (3-NPA) production. METHODS AND RESULTS: To investigate the antifungal mechanism, the effects of TA on the hypha growth, electrical conductivity, hypha morphology, defense-related enzymes, and 3-NPA production of A. arundinis were studied. TA concentrations of 640 and 1280 µg ml-1 exhibited strong antifungal activity against A. arundinis. The results of scanning electron microscopy and transmission electron microscopy showed that the hypha of the A. arundinis was severely deformed after TA treatment, and the cell membrane was blurred and thin, vacuoles were obviously shrunken and smaller, and most of the organelles were decomposed into irregular fragments. The increased electrical conductivity and malondialdehyde content indicated that TA caused peroxidation of unsaturated fatty acids and damaged the structure of the cell membrane. The decrease of intracellular ATPase and succinate dehydrogenase content indicated that TA damaged the function of mitochondria, and participated in the inhibition of respiratory metabolism. In addition, TA significantly reduced 3-NPA production and completely inhibited 3-NPA production at 640 and 1280 µg ml-1. CONCLUSION: TA effectively inhibited both growth of A. arundinis in vitro and 3-NPA production.

Phosphorylation status of CPK28 affects its ubiquitination and protein stability.

Plant innate immunity is tightly regulated. The Arabidopsis thaliana CALCIUM-DEPENDENT PROTEIN KINASE28 (CPK28) functions as a negative immune regulator. We recently demonstrate that CPK28 undergoes ubiquitination that is mediated by two ubiquitin ligases, ARABIDOPSIS TÓXICOS EN LEVADURA31 (ATL31) and ATL6, which results in its proteasomal degradation. CPK28 undergoes both intermolecular autophosphorylation and BIK1-mediated phosphorylation. However, whether the phosphorylation status of CPK28 dictates its ubiquitination and degradation is unknown yet. We used immune response analysis, transient degradation system, ubiquitination assays, co-immunoprecipitation, and other biochemical and genetic approaches to investigate the effect of the phosphorylation status of CPK28 on its degradation mediated by ATL31/6. We found the mutation of Ser318 (a site of both intermolecular autophosphorylation and BIK1-mediated phosphorylation) or a BIK1 phosphorylation site on CPK28 leads to its compromised association with ATL31 and reduced ubiquitination by ATL31. Moreover, we confirm the previous findings that two CPK28s can interact with each other, which likely promotes the intermolecular autophosphorylation. We also show that the phosphorylation status of CPK28 in turn affects its intermolecular association. We demonstrate that the phosphorylation status of CPK28 affects its degradation mediated by ATL31. Our findings reveal a link between phosphorylation of CPK28 and its ubiquitination and degradation.

Biocontrol ability of Bacillus velezensis T9 against Apiospora arundinis causing Apiospora mold on sugarcane.

Sugarcane (Saccharum officinarum L.) may be infected with Apiospora, which can produce the toxin 3-nitropropionic acid (3-NPA) during improper transportation and storage. The consumption of sugarcane that contains 3-NPA can lead to food poisoning. Therefore, this study sought to explore a novel biocontrol agent to prevent and control Apiospora mold. Bacteria were isolated from the soil of healthy sugarcane and identified as Bacillus velezensis T9 through colony morphological, physiological and biochemical characterization and molecular identification. The inhibitory effect of B. velezensis T9 on Apiospora mold on sugarcane was analyzed. Assays of the cell suspension of strain T9 and its cell-free supernatant showed that T9 had significant in vitro antifungal activities against Apiospora arundinis and thus, would be a likely antagonist. Scanning electron microscopy and transmission electron microscopy showed that treatment with T9 significantly distorted the A. arundinis mycelia, perforated the membrane, contracted the vesicles, and decomposed most organelles into irregular fragments. A re-isolation experiment demonstrates the ability of T9 to colonize the sugarcane stems and survive in them. This strain can produce volatile organic compounds (VOCs) that are remarkably strong inhibitors, and it can also form biofilms. Additionally, the cell-free supernatant significantly reduced the ability of A. arundinis to produce 3-NPA and completely inhibited its production at 10%. Therefore, strain T9 is effective at controlling A. arundinis and has the potential for further development as a fungal prevention agent for agricultural products.

Coupled mechanism of enhanced and inhibitory effects of nanoscale zero-valent iron on methane production and antibiotic resistance genes in anaerobic digestion of swine manure.

In this study, the turning point for nanoscale zero-valent iron's (NZVI) promotion and inhibition effects of methane production coupled with the reduction of antibiotic resistance genes (ARGs) was investigated. Adding 150 mmol/L NZVI increased methane production by maximum of 23.8 %, which was due to the chemical reaction producing H2 and enhancement of direct interspecies electron transfer (DIET) by NZVI. NZVI350 dramatically repressed methane generation by 48.0 %, which might be associated with the large quantity of reactive oxygen species (ROS) and excessive H2 inhibiting the functioning of microorganisms. The fate of ARGs was significantly related to daily methane production, indicating that the more methane production finally generated, the less the abundance of ARGs at last left. The reduction of ARGs was enhanced by maximum of 61.0 %, which was attributed to the inhibition of vertical gene transfer (VGT) and horizontal gene transfer (HGT) caused by steric hindrance associated with NZVI corrosion.

Two protein disulfide isomerase subgroups work synergistically in catalyzing oxidative protein folding.

Disulfide bonds play essential roles in the folding of secretory and plasma membrane proteins in the endoplasmic reticulum (ER). In eukaryotes, protein disulfide isomerase (PDI) is an enzyme catalyzing the disulfide bond formation and isomerization in substrates. The Arabidopsis (Arabidopsis thaliana) genome encodes diverse PDIs including structurally distinct subgroups PDI-L and PDI-M/S. It remains unclear how these AtPDIs function to catalyze the correct disulfide formation. We found that one Arabidopsis ER oxidoreductin-1 (Ero1), AtERO1, can interact with multiple PDIs. PDI-L members AtPDI2/5/6 mainly serve as an isomerase, while PDI-M/S members AtPDI9/10/11 are more efficient in accepting oxidizing equivalents from AtERO1 and catalyzing disulfide bond formation. Accordingly, the pdi9/10/11 triple mutant exhibited much stronger inhibition than pdi1/2/5/6 quadruple mutant under dithiothreitol treatment, which caused disruption of disulfide bonds in plant proteins. Furthermore, AtPDI2/5 work synergistically with PDI-M/S members in relaying disulfide bonds from AtERO1 to substrates. Our findings reveal the distinct but overlapping roles played by two structurally different AtPDI subgroups in oxidative protein folding in the ER.

The calcium-dependent protein kinase CPK28 is targeted by the ubiquitin ligases ATL31 and ATL6 for proteasome-mediated degradation to fine-tune immune signaling in Arabidopsis.

Immune responses are triggered when pattern recognition receptors recognize microbial molecular patterns. The Arabidopsis (Arabidopsis thaliana) receptor-like cytoplasmic kinase BOTRYTIS-INDUCED KINASE1 (BIK1) acts as a signaling hub of plant immunity. BIK1 homeostasis is maintained by a regulatory module in which CALCIUM-DEPENDENT PROTEIN KINASE28 (CPK28) regulates BIK1 turnover via the activities of two E3 ligases. Immune-induced alternative splicing of CPK28 attenuates CPK28 function. However, it remained unknown whether CPK28 is under proteasomal control. Here, we demonstrate that CPK28 undergoes ubiquitination and 26S proteasome-mediated degradation, which is enhanced by flagellin treatment. Two closely related ubiquitin ligases, ARABIDOPSIS TÓXICOS EN LEVADURA31 (ATL31) and ATL6, specifically interact with CPK28 at the plasma membrane; this association is enhanced by flagellin elicitation. ATL31/6 directly ubiquitinate CPK28, resulting in its proteasomal degradation. Furthermore, ATL31/6 promotes the stability of BIK1 by mediating CPK28 degradation. Consequently, ATL31/6 positively regulate BIK1-mediated immunity. Our findings reveal another mechanism for attenuating CPK28 function to maintain BIK1 homeostasis and enhance immune responses.

The C-terminal domain of the type III secretion chaperone HpaB contributes to dissociation of chaperone-effector complex in Xanthomonas campestris pv. campestris.

Many animal and plant pathogenic bacteria employ a type three secretion system (T3SS) to deliver type three effector proteins (T3Es) into host cells. Efficient secretion of many T3Es in the plant pathogen Xanthomonas campestris pv. campestris (Xcc) relies on the global chaperone HpaB. However, how the domain of HpaB itself affects effector translocation/secretion is poorly understood. Here, we used genetic and biochemical approaches to identify a novel domain at the C-terminal end of HpaB (amino acid residues 137-160) that contributes to virulence and hypersensitive response (HR). Both in vitro secretion assay and in planta translocation assay showed that the secretion and translocation of T3E proteins depend on the C-terminal region of HpaB. Deletion of the C-terminal region of HpaB did not affect binding to T3Es, self-association or interaction with T3SS components. However, the deletion of C-terminal region sharply reduced the mounts of free T3Es liberated from the complex of HpaB with the T3Es, a reaction catalyzed in an ATP-dependent manner by the T3SS-associated ATPase HrcN. Our findings demonstrate the C-terminal domain of HpaB contributes to disassembly of chaperone-effector complex and reveal a potential molecular mechanism underpinning the involvement of HpaB in secretion of T3Es in Xcc.

Two lytic transglycosylases of Xanthomonas campestris pv. campestris associated with cell separation and type III secretion system, respectively.

The lytic transglycosylases (LTs) are important enzymes that degrade peptidoglycan of the bacterial cell wall and affect many biological functions. We present here that XC_0706 and XC_3001 are annotated as the LTs in Xanthomonas campestris pv. campestris. XC_0706 is associated with virulence and plays a pivotal role in cell division. Mutation on XC_3001 reduced hypersensitive response induction and the translocation of type III effector, but did not affect the function of the type II secretion system. Further studies showed that multiple LTs genes contribute to efficiency of the type III secretory system in X. campestris pv. campestris.

Carbon nanotube-promoted Co-Cu catalyst for highly efficient synthesis of higher alcohols from syngas.

The development of a type of carbon-nanotube-promoted Co-Cu catalyst, which displays excellent performance for highly effective and selective formation of the C(2-4)-oxygenates, especially BuOH and DME, from syngas, is reported.