Lipid mediated plant immunity in susceptible and tolerant soybean cultivars in response to Phytophthora sojae colonization and infection.

BACKGROUND: Soybean is one of the most cultivated crops globally and a staple food for much of the world's population. The annual global crop losses due to infection by Phytophthora sojae is currently estimated at $20B USD, yet we have limited understanding of the role of lipid mediators in the adaptative strategies used by the host plant to limit infection. Since root is the initial site of this infection, we examined the infection process in soybean root infected with Phytophthora sojae using scanning electron microscopy to observe the changes in root morphology and a multi-modal lipidomics approach to investigate how soybean cultivars remodel their lipid mediators to successfully limit infection by Phytophthora sojae. RESULTS: The results reveal the presence of elevated biogenic crystals and more severe damaged cells in the root morphology of the infected susceptible cultivar compared to the infected tolerant cultivars. Furthermore, induced accumulation of stigmasterol was observed in the susceptible cultivar whereas, induced accumulation of phospholipids and glycerolipids occurred in tolerant cultivar. CONCLUSION: The altered lipidome reported in this study suggest diacylglycerol and phosphatidic acid mediated lipid signalling impacting phytosterol anabolism appears to be a strategy used by tolerant soybean cultivars to successfully limit infection and colonization by Phytophthora sojae.

Molecular mechanism for endo-type action of glycoside hydrolase family 55 endo-ß-1,3-glucanase on ß1-3/1-6-glucan.

The glycoside hydrolase family 55 (GH55) includes inverting exo-ß-1,3-glucosidases and endo-ß-1,3-glucanases, acting on laminarin, which is a ß1-3/1-6-glucan consisting of a ß1-3/1-6-linked main chain and ß1-6-linked branches. Despite their different modes of action toward laminarin, endo-ß-1,3-glucanases share with exo-ß-1,3-glucosidases conserved residues that form the dead-end structure of subsite -1. Here, we investigated the mechanism of endo-type action on laminarin by GH55 endo-ß-1,3-glucanase MnLam55A, identified from Microdochium nivale. MnLam55A, like other endo-ß-1,3-glucanases, degraded internal ß-d-glucosidic linkages of laminarin, producing more reducing sugars than the sum of d-glucose and gentiooligosaccharides detected. ß1-3-Glucans lacking ß1-6-linkages in the main chain were not hydrolyzed. NMR analysis of the initial degradation of laminarin revealed that MnLam55A preferentially cleaved the nonreducing terminal ß1-3-linkage of the laminarioligosaccharide moiety at the reducing end side of the main chain ß1-6-linkage. MnLam55A liberates d-glucose from laminaritriose and longer laminarioligosaccharides, but kcat/Km values to laminarioligosaccharides (≤4.21 s-1 mM-1) were much lower than to laminarin (5920 s-1 mM-1). These results indicate that ß-glucan binding to the minus subsites of MnLam55A, including exclusive binding of the gentiobiosyl moiety to subsites -1 and -2, is required for high hydrolytic activity. A crystal structure of MnLam55A, determined at 2.4 Å resolution, showed that MnLam55A adopts an overall structure and catalytic site similar to those of exo-ß-1,3-glucosidases. However, MnLam55A possesses an extended substrate-binding cleft that is expected to form the minus subsites. Sequence comparison suggested that other endo-type enzymes share the extended cleft. The specific hydrolysis of internal linkages in laminarin is presumably common to GH55 endo-ß-1,3-glucanases.

Phyto-oxylipin mediated plant immune response to colonization and infection in the soybean-Phytophthora sojae pathosystem.

Introduction: Food security is a major challenge to sustainably supply food to meet the demands of the ever-growing global population. Crop loss due to pathogens is a major concern to overcoming this global food security challenge. Soybean root and stem rot caused by Phytophthora sojae results in approximately 20B $US crop loss annually. Phyto-oxylipins are metabolites biosynthesized in the plants by oxidative transformation of polyunsaturated fatty acids through an array of diverging metabolic pathways and play an important role in plant development and defense against pathogen colonization and infection. Lipid mediated plant immunity is a very attractive target for developing long term resistance in many plants' disease pathosystem. However, little is known about the phyto-oxylipin's role in the successful strategies used by tolerant soybean cultivar to mitigate Phytophthora sojae infection. Methods: We used scanning electron microscopy to observe the alterations in root morphology and a targeted lipidomics approach using high resolution accurate mass tandem mass spectrometry to assess phyto-oxylipin anabolism at 48 h, 72 h and 96 h post infection. Results and discussion: We observed the presence of biogenic crystals and reinforced epidermal walls in the tolerant cultivar suggesting a mechanism for disease tolerance when compared with susceptible cultivar. Similarly, the unequivocally unique biomarkers implicated in oxylipin mediated plant immunity [10(E),12(Z)-13S-hydroxy-9(Z),11(E),15(Z)-octadecatrienoic acid, (Z)-12,13-dihydroxyoctadec-9-enoic acid, (9Z,11E)-13-Oxo-9,11-octadecadienoic acid, 15(Z)-9-oxo-octadecatrienoic acid, 10(E),12(E)-9-hydroperoxyoctadeca-10,12-dienoic acid, 12-oxophytodienoic acid and (12Z,15Z)-9, 10-dihydroxyoctadeca-12,15-dienoic acid] generated from intact oxidized lipid precursors were upregulated in tolerant soybean cultivar while downregulated in infected susceptible cultivar relative to non-inoculated controls at 48 h, 72 h and 96 h post infection by Phytophthora sojae, suggesting that these molecules may be a critical component of the defense strategies used in tolerant cultivar against Phytophthora sojae infection. Interestingly, microbial originated oxylipins, 12S-hydroperoxy-5(Z),8(Z),10(E),14(Z)-eicosatetraenoic acid and (4Z,7Z,10Z,13Z)-15-[3-[(Z)-pent-2-enyl]oxiran-2-yl]pentadeca-4,7,10,13-tetraenoic acid were upregulated only in infected susceptible cultivar but downregulated in infected tolerant cultivar. These microbial originated oxylipins are capable of modulating plant immune response to enhance virulence. This study demonstrated novel evidence for phyto-oxylipin metabolism in soybean cultivars during pathogen colonization and infection using the Phytophthora sojae-soybean pathosystem. This evidence may have potential applications in further elucidation and resolution of the role of phyto-oxylipin anabolism in soybean tolerance to Phytophthora sojae colonization and infection.

Identification and characterization of extracellular GH3 ß-glucosidase from the pink snow mold fungus, Microdochium nivale.

Glycoside hydrolase family 3 (GH3) ß-glucosidase exists in many filamentous fungi. In phytopathogenic fungi, it is involved in fungal growth and pathogenicity. Microdochium nivale is a severe phytopathogenic fungus of grasses and cereals and is the causal agent of pink snow mold, but its ß-glucosidase has not been identified. In this study, a GH3 ß-glucosidase of M. nivale (MnBG3A) was identified and characterized. Among various p-nitrophenyl ß-glycosides, MnBG3A showed activity on d-glucoside (pNP-Glc) and slight activity on d-xyloside. In the pNP-Glc hydrolysis, substrate inhibition occurred (Kis = 1.6 m m), and d-glucose caused competitive inhibition (Ki = 0.5 m m). MnBG3A acted on ß-glucobioses with ß1-3, -6, -4, and -2 linkages, in descending order of kcat/Km. In contrast, the regioselectivity for newly formed products was limited to ß1-6 linkage. MnBG3A has similar features to those of ß-glucosidases from Aspergillus spp., but higher sensitivity to inhibitory effects.

Recent advances in bio-chemical, molecular and physiological aspects of membrane lipid derivatives in plant pathology.

Plant pathogens pose a significant threat to the food industry and food security accounting for 10-40% crop losses annually on a global scale. Economic losses from plant diseases are estimated at $300B for major food crops and are associated with reduced food availability and accessibility and also high food costs. Although strategies exist to reduce the impact of diseases in plants, many of these introduce harmful chemicals to our food chain. Therefore, it is important to understand and utilize plants' immune systems to control plant pathogens to enable more sustainable agriculture. Lipids are core components of cell membranes and as such are part of the first line of defense against pathogen attack. Recent developments in omics technologies have advanced our understanding of how plant membrane lipid biosynthesis, remodelling and/or signalling modulate plant responses to infection. Currently, there is limited information available in the scientific literature concerning lipid signalling targets and their biochemical and physiological consequences in response to plant pathogens. This review focusses on the functions of membrane lipid derivatives and their involvement in plant responses to pathogens as biotic stressors. We describe major plant defense systems including systemic-acquired resistance, basal resistance, hypersensitivity and the gene-for-gene concept in this context.