β-1,6-Glucan is a functionally crucial polysaccharide of the fungal cell wall, although typically less abundant than β-1,3-glucan and chitin, its content, chain length, and branching vary considerably among species. Structurally, it serves as a covalent cross-linker tethering the external mannoprotein layer to the internal β-1,3-glucan-chitin network, contributing to cell wall integrity and plasticity. Biosynthetically, unlike chitin and β-1,3-glucan, which are synthesized by the plasma membrane-associated synthases, β-1,6-glucan biosynthesis depends on a multi-protein cooperative network spanning the endoplasmic reticulum (ER), Golgi, and cell surface, whose core catalytic machinery remains incompletely defined. Genetic and in vitro reconstitution studies have begun to delineate the contributions of ER-resident proteins (Kre5, Big1, Cwh41/Gls1, Rot2/Gls2, and Cne1), Golgi-localized Kre6/Skn1 family members, and cell-surface components (Kre9, Knh1, Kre1, and Kre11). Functionally, its biological roles are established by two complementary lines of evidence, namely enzymatic digestion by endogenous or exogenous β-1,6-glucanases and inactivation of the biosynthetic machinery. Collectively, these studies show that β-1,6-glucan is essential for cell wall architecture, GPI-anchored protein localization, fungal growth, morphogenesis, and virulence, and acts as a potent immunomodulatory molecule at the fungus–host interface. Elucidating its structure, biosynthesis, and function will advance fungal cell wall biology.

Rice endosperm, the major edible portion of the grain, plays an important role in regulating blood glucose and preventing intestinal diseases by increasing its resistant starch (RS) content. Previous studies have shown that suppressing amylopectin biosynthesis via genome editing can increase RS content. However, the influence of different Waxy (Wx) allelic backgrounds on RS accumulation in edited lines has not been systematically evaluated. In this study, we used glutinous rice Yunan Heixiangnuo (HXN) with a nonfunctional wx allele and indica rice Yixiang 1B (YX1B) with a weak Wxb allele as backgrounds. We simultaneously knocked out SSSIIIa, SBEI, SBEIIa, and SBEIIb using CRISPR/Cas9, and systematically analyzed changes in RS content, rice quality, and yield traits. The results showed that, in the HXN background, multigene knockout did not significantly alter amylose or RS content but largely maintained favorable eating quality. In contrast, in the YX1B background, quadruple-gene knockout lines exhibited an increase in amylose content from 17.7

N-glycosylation, a crucial eukaryotic post-translational modification, has been extensively studied for its significance in the physiology and virulence processes of plant pathogens over the last decade. This review systematically analyzes the functions of N-glycosylation-related endoplasmic reticulum (ER) proteins in regulating plant pathogen processes, including mycelial growth, conidial development, host penetration as well as colonization, pathogenicity, cell wall integrity and host immune evasion. By modifying the structure and function of target proteins, these ER-localized proteins regulate essential developmental events in pathogens while concurrently mediating interactions between pathogens and plants, influencing pathogens’ growth and disease-causing potential. Future research requires the systematic delineation of glycosylation-regulated protein networks by multi-omics integration technologies and the elucidation of their functional processes using molecular genetics methodologies, thereby establishing a robust theoretical foundation for the development of novel biological fungicides.

Fungal cell wall is essential in orchestrating fungal morphogenesis and sensing environmental stimuli. Its dynamic reconstruction involves glycoside hydrolases, particularly β-1,6-glucanase, which is crucial for synthesis of β-1,6-glucan. This glucan functions as a fundamental structural element within the fungal cell wall, covalently linking mannoproteins into the β-1,3-glucan-chitin network. Previously, a highly hydrolytically active β-1,6-glucanase, MoGlu16, was identified in Magnaporthe oryzae. However, its impacts on the pathogenicity of the blast fungus remain unexplored. In this study, to elucidate the detailed biological function of this protein, we generated the ΔMoglu16 mutant strain by knocking out the MoGLU16 gene encoding the β-1,6-glucanase via homologous recombination. Compared to the wild-type strain Guy11, ΔMoglu16 mutant exhibited a significantly slower vegetative growth rate, with a reduction of approximately 52

Xylanases derived from fungi, including phytopathogenic and nonpathogenic fungi, are commonly known to trigger plant immune responses. However, there is limited research on the ability of bacterial-derived xylanases to trigger plant immunity. Here, a novel xylanase named CcXyn was identified from the myxobacterium Cystobacter sp. 0969, which displays broad-spectrum activity against both phytopathogenic fungi and bacteria. CcXyn belongs to the glycoside hydrolases (GH) 11 family and shares a sequence identity of approximately 32.0%-45.0% with fungal xylanases known to trigger plant immune responses. Treatment of Nicotiana benthamiana with purified CcXyn resulted in the induction of hypersensitive response (HR) and defence responses, such as the production of reactive oxygen species (ROS) and upregulation of defence gene expression, ultimately enhancing the resistance of N. benthamiana to Phytophthora nicotianae. These findings indicated that CcXyn functions as a microbe-associated molecular pattern (MAMP) elicitor for plant immune responses, independent of its enzymatic activity. Similar to fungal xylanases, CcXyn was recognized by the NbRXEGL1 receptor on the cell membrane of N. benthamiana. Downstream signalling was shown to be independent of the BAK1 and SOBIR1 co-receptors, indicating the involvement of other co-receptors in signal transduction following CcXyn recognition in N. benthamiana. Moreover, xylanases from other myxobacteria also demonstrated the capacity to trigger plant immune responses in N. benthamiana, indicating that xylanases in myxobacteria are ubiquitous in triggering plant immune functions. This study expands the understanding of xylanases with plant immune response-inducing properties and provides a theoretical basis for potential applications of myxobacteria in biocontrol strategies against phytopathogens.

As high-value chitosan derivatives, chitooligosaccharides (COSs) with biodegradable, biocompatible, nontoxic, antimicrobial, and antioxidant activities have been widely applied in food-related fields. Chitosanases can hydrolyze chitosan to produce COSs. Herein, a chitosanase (LeCho1) from Lentinula edodes was successfully expressed in Escherichia coli and was then purified and characterized. LeCho1 had a low sequence identity with other chitosanases reported from the GH75 family. The recombinant protein showed a molecular mass of 27 kDa on SDS-PAGE. LeCho1 preferentially hydrolyzed chitosan with a high degree of deacetylation (DDA) and exhibited maximal activity (71.88 U/mg) towards 95% DDA chitosan at pH 3.0 and 50 °C. It possessed good stability at pH 2.0–6.0 and temperatures below 45 °C. Its hydrolytic activity was remarkably enhanced by the metal ion Mn2+ at 1 mM, while it was totally inhibited by 1 mM Fe3+ or 10 mM EDTA. Its Km and Vmax values were 0.04 μM and 76.81 μmol·min−1·mg−1, respectively, indicating good substrate affinity. LeCho1 degraded chitosan into COSs with degrees of polymerization (DPs) of 2–5, while it had no action on COSs with DPs of less than 5, revealing its endo-chitosanase activity. This study proved that chitosanase LeCho1 is a promising candidate in the industrial preparation of COSs due to its excellent properties.

As an essential component of the fungal cell wall, β-1,6-glucan has an important role in the growth and development of fungi, but its distribution has not been investigated in Magnaporthe oryzae. Here, a novel β-1,6-glucanase from M. oryzae, MoGlu16, was cloned and expressed in Pichia pastoris. The enzyme was highly active on pustulan, with a specific activity of 219.0 U/mg at pH 5.0 and 50°C, and showed great selectivity for continuous β-1,6-glycosidic bonding polysaccharides. Based on this, β-1,6-glucan was selectively visualized in the vegetative hyphae, conidia and bud tubes of M. oryzae using a hydrolytically inactive GFP-tagged MoGlu16 with point mutations at the catalytic position (His-MoGlu16E236A-Gfp). The spore germination and appressorium formation were significantly inhibited after incubation of 105/ml conidia with 0.03 μg/μl MoGlu16. Mycelia treated with MoGlu16 produced reactive oxygen species and triggered the cell wall integrity pathway, increasing the expression levels of genes involved in cell wall polysaccharide synthesis. These results revealed that MoGlu16 participated in the remodeling of cell wall in M. oryzae, laying a foundation for the analysis of cell wall structure.

β-1,3-glucanases can degrade β-1,3-glucoside bonds in β-glucan which is the main cell-wall component of most of fungi, and have the crucial application potential in plant protection and food processing. Herein, a β-1,3-glucanase FlGluA from Flavobacterium sp. NAU1659 composed of 333 amino acids with a predicted molecular mass of 36.6 kDa was expressed in Escherichia coli BL21, purified and characterized. The deduced amino acid sequence of FlGluA showed the high identity with the β-1,3-glucanase belonging to glycoside hydrolase (GH) family 16. Enzymological characterization indicated FlGluA had the highest activity on zymosan A, with a specific activity of 3.87 U/mg, followed by curdlan (1.16 U/mg) and pachymaran (0.88 U/mg). It exhibited optimal catalytic activity at the pH 5.0 and 40 °C, and was stable when placed at 4 °C for 12 h in the range of pH 3.0–8.0 or at a temperature below 50 °C for 3 h. Its catalytic activity was enhanced by approximately 36 % in the presence of 1 mM Cr3+. The detection of thin-layer chromatography and mass spectrometry showed FlGluA hydrolyzed zymosan A mainly to glucose and disaccharide, and trace amounts of tetrasaccharide and pentasaccharide, however, it had no action on laminaribiose, indicating its endo-β-1,3-glucanase activity. The mycelium growth of F. oxysporum treated by FlGluA was inhibited, with approximately 37 % of inhibition rate, revealing the potential antifungal activity of the enzyme. These results revealed the hydrolytic properties and biocontrol activity of FlGluA, laying a crucial foundation for its potential application in agriculture and industry.

A novel amylase AmyFlA from Flavobacterium sp. NAU1659, AmyFlA, was cloned and expressed in Esherichia coli . Based on phylogenetic and functional analysis, it was identified as a novel member of the subfamily GH13_46, sharing high sequence identity. The protein was predicted to consist of 620 amino acids, with a putative signal peptide of 25 amino acids. The enzyme was able to hydrolyze soluble starch with a specific activity of 352.97 U/mg at 50 °C in 50 mM phosphate buffer (pH 6.0). The K m and V max values of AmyFlA were respectively 3.15 mg/ml and 566.36 µmol·ml −1 ·min −1 under optimal conditions. Its activity towards starch was enhanced by 63% in the presence of 1 mM Ca 2+ , indicating that AmyFlA was a Ca 2+ -dependent amylase. Compared to the reported maltogenic amylases, AmyFlA produced a lower variety of intermediate oligosaccharides at the start of the reaction so that the product mixture contained a higher proportion of maltose. These results indicate that AmyFlA may be potential application value in the production of high-maltose syrup.

(3-1,3-glucanases can degrade (3-1,3-glucoside bonds in (3-glucan which is the main cell-wall component of most of fungi, and have the crucial application potential in plant protection and food processing. Herein, a (3-1,3-glucanase FlGluA from Flavobacterium sp. NAU1659 composed of 333 amino acids with a predicted molecular mass of 36.6 kDa was expressed in Escherichia coli BL21, purified and characterized. The deduced amino acid sequence of FlGluA showed the high identity with the (3-1,3-glucanase belonging to glycoside hydrolase (GH) family 16. Enzymological characterization indicated FlGluA had the highest activity on zymosan A, with a specific activity of 3.87 U/mg, followed by curdlan (1.16 U/mg) and pachymaran (0.88 U/mg). It exhibited optimal catalytic activity at the pH 5.0 and 40 degrees C, and was stable when placed at 4 degrees C for 12 h in the range of pH 3.0-8.0 or at a temperature below 50 degrees C for 3 h. Its catalytic activity was enhanced by approximately 36 % in the presence of 1 mM Cr3+. The detection of thin-layer chromatography and mass spectrometry showed FlGluA hydrolyzed zymosan A mainly to glucose and disaccharide, and trace amounts of tetrasaccharide and pentasaccharide, however, it had no action on laminaribiose, indicating its endo-(3-1,3-glucanase activity. The mycelium growth of F. oxysporum treated by FlGluA was inhibited, with approximately 37 % of inhibition rate, revealing the potential antifungal activity of the enzyme. These results revealed the hydrolytic properties and biocontrol activity of FlGluA, laying a crucial foundation for its potential application in agriculture and industry.

Chitosanases hydrolyze chitosan into chitooligosaccharides (COSs) with various biological activities, which are widely employed in many areas including plant disease management. In this study, the novel chitosanase AqCsn1 belonging to the glycoside hydrolase family 46 (GH46) was cloned from Aquabacterium sp. A7-Y and heterologously expressed in Escherichia coli BL21 (DE3). AqCsn1 displayed the highest hydrolytic activity towards chitosan with 95% degree of deacetylation at 40 °C and pH 5.0, with a specific activity of 13.18 U/mg. Product analysis showed that AqCsn1 hydrolyzed chitosan into (GlcN)2 and (GlcN)3 as the main products, demonstrating an endo-type cleavage pattern. Evaluation of antagonistic activity showed that the hydrolysis products of AqCsn1 suppress the mycelial growth of Magnaporthe oryzae and Phytophthora sojae in a concentration-dependent manner, and the inhibition rate of P. sojae reached 39.82% at a concentration of 8 g/L. Our study demonstrates that AqCsn1 and hydrolysis products with a low degree of polymerization might have potential applications in the biological control of agricultural diseases.

褐飞虱发生危害范围十分广泛,是水稻上的重要害虫,具有隐蔽性、 暴发性和迁飞性等特点.2005—2021年杭州年均产量损失5893 t,并数次大规模暴发,严重威胁当地的水稻生产安全.本研究分析其历年发生特点,对其主治药剂烯啶虫胺·吡蚜酮、呋虫胺和三氟苯嘧啶等化学药剂进行了应急防效试验.研究表明,防治褐飞虱药剂可选用烯啶·吡蚜酮、三氟苯嘧啶等药剂,使用时注意轮换用药,在大暴发时可选用毒死蜱+吡蚜酮压低虫口密度.通过科学使用防治技术控制杭州地区的褐飞虱发生为害,保障粮食生产安全,实现虫口夺粮.

Plant-associated nitrogen-fixing microorganisms (diazotrophs) are essential to host nutrient acquisition, productivity and health, but how host growth affects the succession characteristics of crop diazotrophic communities is still poorly understood. Here, Illumina sequencing of DNA- and RNA-derived nifH genes was employed to investigate the dynamics of total and active diazotrophic communities across rhizosphere soil and rice roots under four fertilization regimes during three growth periods (tillering, heading and mature stages) of rice in 2015 and 2016. Our results indicated that 71.9–77.2% of the operational taxonomic units (OTUs) were both detected at the DNA and RNA levels. According to the nonmetric multidimensional scaling ordinations of Bray–Curtis distances, the variations in community composition of active rhizosphere diazotrophs were greater than those of total rhizosphere diazotrophs. The community composition (β-diversity) of total and active root-associated diazotrophs was shaped predominantly by microhabitat (niche; R2 ≥ 0.959, p < 0.001), followed by growth period (R2 ≥ 0.15, p < 0.001). The growth period had a stronger effect on endophytic diazotrophs than on rhizosphere diazotrophs. From the tillering stage to the heading stage, the α-diversity indices (Chao1, Shannon and phylogenetic diversity) and network topological parameters (edge numbers, average clustering coefficient and average degree values) of total endophytic diazotrophic communities increased. The proportions of OTUs shared by the total rhizosphere and endophytic diazotrophs in rhizosphere diazotrophs gradually increased during rice growth. Moreover, total diazotrophic α-diversity and network complexity decreased from rhizosphere soil to roots. Collectively, compared with total diazotrophic communities, active diazotrophic communities were better indicators of biological response to environmental changes. The host microhabitat profoundly drove the temporal dynamics of total and active root-associated diazotrophic communities, followed by the plant growth period.

Dietary starch with an increased content of resistant starch (RS) has the potential to reduce the prevalence of diabetes, obesity, and cardiovascular diseases. Here, an efficient glycogen branching enzyme, CcGBE, from Corallococcus sp. strain EGB was identified, and its relevant properties, including potential application in the preparation of modified starch, were evaluated. The purified CcGBE exhibited a maximal specific activity of approximately 20,000 U/mg using cassava starch as the optimal substrate. The content of α-1,6-glucosidic bonds in CcGBE-modified cassava starch increased from 2.9 to 13.2%. Meanwhile, both the average chain length (CL) of CcGBE-modified starch and the blue value of the color complex formed by starch and iodine initially increased and then decreased, indicating that a new CL transfer mode was reported. Perforated small starch granules were released after CcGBE treatment, and a time-dependent decrease in the retrogradation enthalpy (ΔHr) of cassava starch indicated that CcGBE inhibited the long-term retrogradation of starch. Moreover, the RS content and cold water solubility (CWS) of CcGBE-modified starch increased from 3.3 to 12.8% and from 23.1 to 93.8%, respectively. These findings indicate the application potential of CcGBE for the preparation of modified starch with increased RS and CWS.

Aim To investigate the broad-spectrum antifungal activity of Burkholderia sp. BV6, that is isolated from rice roots and its biocontrol potential against rice blast caused by Magnaporthe oryzae. Methods and Results We evaluated the ability to isolate BV6 in the biological control of rice blast disease and investigated its antifungal mechanisms. BV6 strongly inhibited the hyphal growth of M. oryzae Guy11 and other plant pathogenic fungi, and pot experiments showed that BV6 significantly decreases the disease index of rice blast from 47.5 to 24.6. The secreted small-molecule secondary metabolites were regarded as weapons during the antifungal process by inhibiting the germination of M. oryzae conidia and mycelial growth, and thereby prevent the following infection. Liquid chromatography-mass spectrometry analysis of the metabolites from the supernatant of isolate BV6 showed that the antifungal weapons of isolate BV6 are novel, small, molecular hydrophilic compounds that are different from reported antifungal compounds. Conclusions The isolate BV6 inhibits the M. oryzae infection by the production of small-molecule antifungal compounds. Significance and Impact of the Study The current study discovers the role of the Burkholderia sp. BV6 in the biocontrol of plant pathogenic fungi. Therefore, isolate BV6 is a potential candidate for developing a microbial formulation for the biocontrol of the most common disease of rice blast.

Fungal cell wall synthesizing enzymes or remodeling enzymes represent key factors for the interaction of plant pathogen and antifungal agents, which are regarded as potential biocontrol agents. In this study, a novel endo-β-1,3-glucanase from Magnaporthe oryzae was expressed and characterized. The expression of MoGluB was significantly upregulated after 2 days of liquid culture and 48 h after infection, indicating that it may be involved in cell wall reconstitution. Purified MoGluB exhibited high activity on insoluble β-glucans, with a specific activity of 8.18 U/mg toward yeast glucan at pH 9.0 and 50 °C. MoGluB hydrolyzed pachymaran and yeast glucan into oligosaccharides dominated by laminaripentaose, suggesting that it is an endo-β-1,3-glucanase. Incubation of 8 μg of MoGluB with 106 spores/mL resulted in the inhibition of conidial germination and appressorium formation of M. oryzae, illustrating effective biocontrol activity. Hydrolysates of pachymaran induced the expression of defense genes restricting M. oryzae infection in rice plants, indicating an immunostimulatory effect of MoGluB hydrolysates.

Enzymes that degrade fungal cell walls and the resulting oligosaccharides are promising weapons to combat plant fungal disease. In this study, we identified a novel endo-chitosanase, AqCoA, from Aquabacterium sp. A7-Y. The enzyme showed a specific activity of 18 U/mg toward 95% deacetylated chitosan at pH 5.0 and 40 °C. AqCoA also showed activity toward sodium carboxymethylcellulose, indicating substrate promiscuity. AqCoA hydrolyzed chitosan into chitooligosaccharides (CoA-COSs) with degrees of polymerization (DPs) of 3-5 but showed no activity toward CoA-COSs with DPs <6, indicating an endo-type activity. At 2.5 μg/mL, AqCoA inhibited appressorium formation of Magnaporthe oryzae; the produced CoA-COSs also inhibited the growth of M. oryzae and Fusarium oxysporum. Furthermore, CoA-COSs acted as immune elicitors in rice by inducing the reactive oxygen species burst and the expression of defense genes. These results demonstrated that AqCoA and its resulting CoA-COSs might be effective tools for protecting plants against pathogenic fungi.

In our previous study, the chitosanase AqCoA and the chitooligosaccharides it produced were found to exhibit significant protective effects against fungal diseases. In this study, we enhanced the expression of AqCoA using the novel pMC-GAP that enables stable transformation of Escherichia coli, and built an integrated model based on the gene copy number, molecular chaperones, and protein production of AqCoA. In terms of gene dosage, the highest hydrolase activity was 0.32 U/ml in the strain with four copies, which was 1.78-fold higher than that of the strain with only one copy (0.18 U/ml). In addition, we found the chaperones such as PDI, ERO1, HAC1, YDJ1, SSE1, SSA4, and SSO2 improved protein expression. Furthermore, the PDI/ERO1, SSA4/SSE1, and YDJ1/SSO2 pairs synergistically increased the expression levels by 61%, 31%, and 42%, respectively. Finally, we investigated the combined effects of gene copy numbers and molecular chaperones on protein expression. The highest activity reached 2.32 U/ml in the strain with six integrated molecular chaperone expression cassettes and sixteen copies of the target gene, which was 13-fold higher than that of the control strain with only one copy (GAP-1AqCoA). Combined optimization of gene dosage and molecular chaperone combinations significantly increased the expression level of AqCoA, providing a powerful strategy to improve the expression of other heterologous proteins in P. pastoris.

In our previous study, the chitosanase Aq CoA and its products chitooligosaccharides exhibited significant application in fungal disease protection. In this study, to enhance the expression of Aq CoA, we obtained various strains with multi-copy by a novel plasmid pMC-GAP with stable transformation ability in Pichia pastoris and built an integrated model combining the gene copy number, the chaperones and protein production of Aq CoA. In terms of gene dosage, the highest enzyme activity was 0.32 U/ml in the strain with four copies, which was 1.78-fold higher than that in strain with only one copy (0.18 U/ml). In addition, we found the chaperone like PDI, ERO1, HAC1, YDJ1, SSE1, SSA4 and SSO2 improved protein expression. Furthermore, the PDI/ERO1, SSA4/SSE1 and YDJ1/SSO2 pairs synergistically increased by 61%, 31% and 42% in expression levels of the strain GAP-1 Aq CoA. Finally, we investigated the effect co-expression of gene copy and chaperones on protein expression. The maximum activity reached 2.319 U/ml by the strain with six chaperones intergrant plus sixteen copies, which was 13-fold higher than that by the control strain with only one copy (GAP-1AqCoA). Co-expression of gene dosage and chaperones significantly enhanced expression levels of Aq CoA, which presented a powerful tool to improve foreign protein expression.