Glutamine synthetase gene PpGS1.1 negatively regulates the powdery mildew resistance in Kentucky bluegrass

Abstract

Excessive nitrogen (N) application may induce powdery mildew (PM) in perennial grasses, but the resistance mechanisms to PM remain unclear. This study evaluated the physiological and molecular mechanisms of PM resistance affected by N supplies in Kentucky bluegrass (Poa pratensis L.). Cultivar ‘Bluemoon’ (N tolerant) and ‘Balin’ (N sensitive) were treated with low N (0.5 mM), normal N (15 mM), and high N (30 mM) for 21 d in a greenhouse. With increasing N levels, the disease growth was more severe in ‘Balin’ than in ‘Bluemoon’. RNA-seq and weighted gene coexpression network analysis revealed that the PpGS1.1 gene encoding glutamine synthetase was a potential hub gene for PM resistance after comparisons across cultivars and N treatments. The N metabolism pathway was connected with the plant–pathogen interaction pathway via PpGS1.1. The expression of PpGS1.1 in rice protoplasts indicated that the protein was located in the nucleus and cytoplasm. Overexpression of PpGS1.1 in wild-type Kentucky bluegrass increased carbon and N contents, and the transgenic plants became more susceptible to PM with a lower wax density. The most differentially expressed genes (DEGs) for N metabolism were upregulated and DEGs for fatty acid metabolism pathway were downregulated in the overexpression lines. The results elucidated mechanisms of PM resistance in relation to N metabolism in Kentucky bluegrass.

References

[1] Sun X, Guo Z, Jiang Y et al. Differential metabolomic responses of Kentucky bluegrass cultivars to low nitrogen stress. Front Plant Sci. 2022; 12: 808772-2.

[2] Zhu M, Ji J, Shi W et al. Occurrence of powdery mildew caused by Blumeria graminis f. sp. poae on Poa pratensis in China. Plant Dis. 2021; 105: 1212.

[3] Meyer WA, Hoffman L, Bonos SA . Breeding disease-resistant cool-season turfgrass cultivars for the United States. Plant Dis. 1982; 66: 341.

[4] Czembor E, Feuerstein U, Zurek G . Powdery mildew resistance in Kentucky bluegrass ecotypes from Poland. Plant Breed Seed Sci. 2001; 45: 21-31.

[5] He H, Liu R, Ma P et al. Characterization of Pm68, a new powdery mildew resistance gene on chromosome 2BS of Greek durum wheat TRI 1796. Theor Appl Genet. 2021; 134: 53-62.

[6] Moore JW, Herrera-Foessel S, Lan C et al. A recently evolved hexose transporter variant confers resistance to multiple pathogens in wheat. Nat Genet. 2015; 47: 1494-8.

[7] Liu Y, Xin J, Liu L et al. A temporal gene expression map of chrysanthemum leaves infected with Alternaria alternata reveals different stages of defense mechanisms. Hortic Res. 2020; 7: 23.

[8] Consonni C, Humphry ME, Hartmann HA et al. Conserved requirement for a plant host cell protein in powdery mildew pathogenesis. Nat Genet. 2006; 38: 716-20.

[9] Hu Y, Cheng Y, Yu X et al. Overexpression of two CDPKs from wild Chinese grapevine enhances powdery mildew resistance in Vitis vinifera and Arabidopsis. New Phytol. 2021; 230: 2029-46.

[10] Halterman D, Zhou F, Wei F et al. The MLA6 coiled-coil, NBS-LRR protein confers AvrMla6-dependent resistance specificity to Blumeria graminis f. sp. hordei in barley and wheat. Plant J. 2001; 25: 335-48.

[11] Wang W, Wen Y, Berkey R et al. Specific targeting of the Arabidopsis resistance protein RPW8.2 to the interfacial membrane encasing the fungal haustorium renders broad-spectrum resistance to powdery mildew. Plant Cell. 2009; 21: 2898-913.

[12] Li Y, Zhang Y, Wang QX et al. RESISTANCE TO POWDERY MILDEW 8.1 boosts pattern-triggered immunity against multiple pathogens in Arabidopsis and rice. Plant Biotechnol J. 2018; 16: 428-41.

[13] Dangl JL, Horvath DM, Staskawicz BJ . Pivoting the plant immune system from dissection to deployment. Science. 2013; 341: 746-51.

[14] Mang HG, Laluk KA, Parsons EP et al. The Arabidopsis RESURRECTION1 gene regulates a novel antagonistic interaction in plant defense to biotrophs and necrotrophs. Plant Physiol. 2009; 151: 290-305.

[15] Zabka V, Stangl M, Bringmann G et al. Host surface properties affect prepenetration processes in the barley powdery mildew fungus. New Phytol. 2008; 177: 251-63.

[16] Fauth M, Schweizer P, Buchala A et al. Cutin monomers and surface wax constituents elicit H₂O₂ in conditioned cucumber hypocotyl segments and enhance the activity of other H₂O₂ elicitors1 . Plant Physiol. 1998; 117: 1373-80.

[17] Mösinge PSAJ . Induction of resistance in barley against Erysiphe graminis f.sp. hordei by free cutin monomers. Physiol Mol Plant Pathol. 1996; 49: 103-20.

[18] Raffaele S, Vailleau F, Léger A et al. A MYB transcription factor regulates very-long-chain fatty acid biosynthesis for activation of the hypersensitive cell death response in Arabidopsis. Plant Cell. 2008; 20: 752-67.

[19] Xue Y, Jiang J, Yang X et al. Genome-wide mining and comparative analysis of fatty acid elongase gene family in Brassica napus and its progenitors. Gene. 2020; 747: 144674.

[20] Wang D, Ni Y, Liao L et al. Poa pratensis ECERIFERUM1 (PpCER1) is involved in wax alkane biosynthesis and plant drought tolerance. Plant Physiol Biochem. 2021; 159: 312-21.

[21] Liu K, Niu Y, Konishi M et al. Discovery of nitrate-CPK-NLP signalling in central nutrient-growth networks. Nature. 2017; 545: 311-6.

[22] Jiang Y, Wang W, Xie Q et al. Plants transfer lipids to sustain colonization by mutualistic mycorrhizal and parasitic fungi. Science. 2017; 356: 1172-5.

[23] Lv G, Ying X, Yayi T et al. Effects of nitrogen and phosphorus limitation on fatty acid contents in aspergillus oryzae. Front Microbiol. 2021; 12: 739569.

[24] Zapletalová A, Ducsay L, Varga L et al. Influence of nitrogen nutrition on fatty acids in oilseed rape (Brassica napus L.). Plants (Basel). 2022; 11: 44.

[25] Zheljazkov VD, Vick B, Ebelhar MW et al. Nitrogen applications modify seed and oil yields and fatty acid composition of winter mustard. Ind Crop Prod. 2012; 36: 28-32.

[26] Ding S, Shao X, Li J et al. Nitrogen forms and metabolism affect plant defence to foliar and root pathogens in tomato. Plant Cell Environ. 2021; 44: 1596-610.

[27] Baba VY, Braghini MT, Santos TB et al. Transcriptional patterns of Coffea arabica L. nitrate reductase, glutamine and asparagine synthetase genes are modulated under nitrogen suppression and coffee leaf rust. PeerJ. 2020; 8: e8320.

[28] Chen Y, Zhang F, Tang L et al. Wheat powdery mildew and foliar N concentrations as influenced by N fertilization and belowground interactions with intercropped faba bean. Plant Soil. 2007; 291: 1-13.

[29] Tavernier V, Cadiou S, Pageau K et al. The plant nitrogen mobilization promoted by Colletotrichum lindemuthianum in Phaseolus leaves depends on fungus pathogenicity. J Exp Bot. 2007; 58: 3351-60.

[30] Pageau K, Reisdorf-Cren M, Morot-Gaudry J et al. The two senescence-related markers, GS1 (cytosolic glutamine synthetase) and GDH (glutamate dehydrogenase), involved in nitrogen mobilization, are differentially regulated during pathogen attack and by stress hormones and reactive oxygen species in Nicotiana tabacum L. leaves. J Exp Bot. 2006; 57: 547-57.

[31] Olea F, Pérez-García A, Cantón FR et al. Up-regulation and localization of asparagine synthetase in tomato leaves infected by the bacterial pathogen pseudomonas syringae. Plant Cell Physiol. 2004; 45: 770-80.

[32] Sun X, Zheng Q, Xiong L et al. Nitrogen assimilation and gene regulation of two Kentucky bluegrass cultivars differing in response to nitrate supply. Sci Hortic. 2021; 288: 110315.

[33] Urriola J, Rathore KS . Overexpression of a glutamine synthetase gene affects growth and development in sorghum. Transgenic Res. 2015; 24: 397-407.

[34] Bian S, Jiang Y . Reactive oxygen species, antioxidant enzyme activities and gene expression patterns in leaves and roots of Kentucky bluegrass in response to drought stress and recovery. Sci Hortic. 2009; 120: 264-70.

[35] Zhao X, Shi W . Expression analysis of the glutamine synthetase and glutamate synthase gene families in young rice (Oryza sativa) seedlings. Plant Sci. 2006; 170: 748-54.

[36] Li S, Lin D, Zhang Y et al. Genome-edited powdery mildew resistance in wheat without growth penalties. Nature. 2022; 602: 455-60.

[37] Toyota M, Spencer D, Sawai-Toyota S et al. Glutamate triggers long-distance, calcium-based plant defense signaling. Science. 2018; 361: 1112-5.

[38] Steffens B, Sauter M . Epidermal cell death in rice is confined to cells with a distinct molecular identity and is mediated by ethylene and H₂O₂ through an autoamplified signal pathway . Plant Cell. 2009; 21: 184-96.

[39] Takahashi F, Mizoguchi T, Yoshida R et al. Calmodulin-dependent activation of MAP kinase for ROS homeostasis in Arabidopsis. Mol Cell. 2011; 41: 649-60.

[40] Todd J, Post-Beittenmiller D, Jaworski JG . KCS1 encodes a fatty acid elongase 3-ketoacyl-CoA synthase affecting wax biosynthesis in Arabidopsis thaliana. Plant J. 1999; 17: 119-30.

[41] Garnier E, Vancaeyzeele S . Carbon and nitrogen content of congeneric annual and perennial grass species: relationships with growth. Plant Cell Environ. 1994; 17: 399-407.

[42] Högberg P, Granström A, Johansson T et al. Plant nitrate reductase activity as an indicator of availability of nitrate in forest soils. Can J For Res. 1986; 16: 1165-9.

[43] Joy KW, Hageman RH . The purification and properties of nitrite reductase from higher plants, and its dependence on ferredoxin. Biochem J. 1966; 100: 263-73.

[44] Oaks A, Stulen I, Jones K et al. Enzymes of nitrogen assimilation in maize roots. Planta. 1980; 148: 477-84.

[45] Lin CC, Kao CH . Disturbed ammonium assimilation is associated with growth inhibition of roots in rice seedlings caused by NaCl. Plant Growth Regul. 1996; 18: 233-8.

[46] Cakmak I, Horst WJ . Horst effect of aluminium on lipid peroxidation, siiperoxide dismutase, catalase, and peroxidase activities in root tips of soybean (Glycine max). Physiol Plant. 1991; 83: 463-8.

[47] Tian M, Gu Q, Zhu M . The involvement of hydrogen peroxide and antioxidant enzymes in the process of shoot organogenesis of strawberry callus. Plant Sci. 2003; 165: 701-7.

[48] Kwak S, Kim S-K, Lee M-S et al. Acidic peroxidases from suspension-cultures of sweet potato. Phytochemistry. 1995; 39: 981-4.

[49] Havir EA, McHale NA . Enhanced-peroxidatic activity in specific catalase isozymes of tobacco, barley, and maize. Plant Physiol. 1989; 91: 812-5.

[50] Grabherr MG, Haas BJ, Yassour M et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol. 2011; 29: 644-52.

[51] Li B, Dewey CN . RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics. 2011; 12: 323-3.

[52] Anders S, Huber W . Differential expression analysis for sequence count data. Genome Biol. 2010; 11: R106.

[53] Zhang B, Horvath S . A general framework for weighted gene co-expression network analysis. Stat Appl Genet Mol Biol. 2005; 4: 17.

[54] Shannon P, Markiel A, Ozier O et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003; 13: 2498-504.

[55] Letunic I, Bork P . 20 years of the SMART protein domain annotation resource. Nucleic Acids Res. 2018; 46: D493-6.

[56] Chang S, Puryear J, Cairney J . A simple and efficient method for isolating RNA from pine trees. Plant Mol Biol Report. 1993; 11: 113-6.

[57] de Kochko A, Hamon S . A rapid and efficient method for the isolation of restrictable total DNA from plants of the genus Abelmoschus. Plant Mol Biol Report. 1990; 8: 3-7.

[58] Huang CX, Canny MJ, Oates K et al. Planing frozen hydrated plant specimens for SEM observation and EDX microanalysis. Microsc Res Tech. 1994; 28: 67-74.

[59] Emanuelsson O, Nielsen H, Brunak S et al. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol. 2000; 300: 1005-16.

[60] Chen C, Chen H, Zhang Y et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant. 2020; 13: 1194-202.