An Aminobutyric Acid Transaminase in Zea mays Interacts With Rhizoctonia solani Cellulase to Participate in Disease Resistance.

Corn sheath blight, caused by AG1-IA, a fusion group of Rhizoctonia solani, which acts as a kind of necrotrophic fungal pathogen, poses a global threat to the production of Zea mays. Although cellulase plays a crucial role in R. solani infections, how plants respond to it is still poorly understood. In this study, we identified a gamma-aminobutyric acid transaminase (GABA-T), ZmGABA-T, in Z. mays that interacts with a cell wall-degrading enzyme (CWDE), EG1, in the cell membrane, using yeast two-hybrid assay, co-immunoprecipitation (Co-IP), and bimolecular fluorescence complementation assays. We found that the combination of EG1 and ZmGABA-T suppressed the allergic necrosis induced by EG1. We also found that the substrate of GABA-T-GABA, can inhibit the transcription of EG1. Transient expression of ZmGABA-T inhibited R. solani infection in Nicotiana benthamiana. The homolog in Oryza sativa, OsGABA-T, could also interact with EG1 to suppress the allergic necrosis induced by EG1. The OsGABA-T knocked out plants displayed enhanced susceptibility to R. solani and showed larger lesions. In conclusion, our results suggest that ZmGABA-T inhibits allergic necrosis induced by EG1 based on the combination with EG1, producing resistance to R. solani infection.

Oxidative cleavage of cellulose in the horse gut.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) belonging to the auxiliary activity 9 family (AA9) are widely found in aerobic fungi. These enzymes are O2-dependent copper oxidoreductases that catalyze the oxidative cleavage of cellulose. However, studies that have investigated AA9 LPMOs of aerobic fungi in the herbivore gut are scare. To date, whether oxidative cleavage of cellulose occurs in the herbivore gut is unknown. RESULTS: We report for the first time experimental evidence that AA9 LPMOs from aerobic thermophilic fungi catalyze the oxidative cleavage of cellulose present in the horse gut to C1-oxidized cellulose and C1- and C4-oxidized cello-oligosaccharides. We isolated and identified three thermophilic fungi and measured their growth and AA9 LPMO expression at 37 °C in vitro. We also assessed the expression and the presence of AA9 LPMOs from thermophilic fungi in situ. Finally, we used two recombinant AA9 LPMOs and a native AA9 LPMO from thermophilic fungi to cleave cellulose to yield C1-oxidized products at 37 °C in vitro. CONCLUSIONS: The oxidative cleavage of cellulose occurs in the horse gut. This finding will broaden the known the biological functions of the ubiquitous LPMOs and aid in determining biological significance of aerobic thermophilic fungi.

Pathogen-Associated Molecular Pattern Active Sites of GH45 Endoglucanohydrolase from Rhizoctonia solani.

A 207-amino-acid residue endoglucanohydrolase (EG1) belonging to the glycoside hydrolase 45 (GH45) from Rhizoctonia solani acts as a pathogen-associated molecular pattern (PAMP). However, the mechanism of EG1 inducing plant immunity is unclear. Here, we found that EG1 contains two domains related to its PAMP function. Transient expression showed that EG1-1, the mutation deleting 60 amino acid residues from the N-terminal, still reserved the PAMP function. Further truncation of EG1-1 obtained two truncating mutations: EG1-2, deleting seven amino acid residues from the N-terminal of EG1-1 (SPWAVND), and EG1-3, deleting five amino acid residues from the C-terminal of EG1-1 (GCSRK). Transient expression showed that the two truncating mutations EG1-2 and EG1-3 all lost the PAMP function. Site-directed mutagenesis of EG1-1 showed that the three amino acid residues (P, W, and D) in the region SPWAVND and the two amino acid residues (C and R) in the region GCSRK were involved in the PAMP function. The homology model showed that the two regions were located at a surface on the EG1 and structurally independent. These results demonstrate that there are two functional regions for the plant immune function of the EG1 released by R. solani, and the two functional regions are independent of each other.

Polysaccharide monooxygenase-catalyzed oxidation of cellulose to glucuronic acid-containing cello-oligosaccharides.

BACKGROUND: Polysaccharide monooxygenases (PMOs) play an important role in the enzymatic degradation of cellulose. They have been demonstrated to able to C6-oxidize cellulose to produce C6-hexodialdoses. However, the biological function of C6 oxidation of PMOs remains unknown. In particular, it is unclear whether C6-hexodialdoses can be further oxidized to uronic acid (glucuronic acid-containing oligosaccharides). RESULTS: A PMO gene, Hipmo1, was isolated from Humicola insolens and expressed in Pichia pastoris. This PMO (HiPMO1), belonging to the auxiliary activity 9 (AA9) family, was shown to able to cleave cellulose to yield non-oxidized and oxidized cello-oligosaccharides. The enzyme oxidizes C6 positions in cellulose to form glucuronic acid-containing cello-oligosaccharides, followed by hydrolysis with beta-glucosidase and beta-glucuronidase to yield glucose, glucuronic acid, and saccharic acid. This indicates that HiPMO1 can catalyze C6 oxidation of hydroxyl groups of cellulose to carboxylic groups. CONCLUSIONS: HiPMO1 oxidizes C6 of cellulose to form glucuronic acid-containing cello-oligosaccharides followed by hydrolysis with beta-glucosidase and beta-glucuronidase to yield glucose, glucuronic acid, and saccharic acid, and even possibly by beta-eliminative cleavage to produce unsaturated cello-oligosaccharides. This study provides a new mechanism for cellulose cleavage by C6 oxidation of HiPMO1.