CRISPR/Cas9-based generation of mlo mutants for allelic complementation experiments to elucidate MLO function in barley

Springer Science and Business Media LLC - 2023
Hina Koide1, Hiroshi Hisano2, Takashi Yaeno1,3
1Department of Agriculture, Ehime University, Matsuyama, Japan
2Institute of Plant Science and Resources, Okayama University, Kurashiki, Japan
3Center for Development of Hulless Barley, Ehime University, Matsuyama, Japan

Tóm tắt

Barley (Hordeum vulgare) mildew locus o (mlo) mutants exhibit strong resistance to penetration by the powdery mildew fungus Blumeria graminis f. sp. hordei. MLO, a seven-transmembrane protein localized at the plasma membrane is thought to be involved in intracellular calcium signaling. However, its molecular function and the mechanism by which mlo mutations confer resistance to penetration by the fungus remain poorly understood. A large number of mlo alleles with different amino acid substitutions at each intracellular loop have been found in various cultivars. However, it is difficult to analyze how each amino acid is involved in penetration resistance by comparing these cultivars because they differ substantially in their genetic background and in the presence or absence of resistance genes recognizing avirulence factors from the pathogen. In this study, we used a CRISPR/Cas9-mediated genetic modification system to generate mlo mutants in the transformable cultivar Golden Promise to enable complementation experiments with the aim of elucidating the molecular function of MLO. An mlo mutant with a thymine insertion in the second exon and penetration resistance to B. graminis f. sp. hordei was obtained. Susceptibility was restored in cells in which the MLO-mCherry gene was introduced using particle bombardment, indicating that this mlo mutant could be a useful genetic tool for complementation experiments using transgenes expressing a variety of mlo alleles.

Từ khóa


Tài liệu tham khảo

Acevedo-Garcia J, Spencer D, Thieron H, Reinstädler A, Hammond-Kosack K, Phillips AL, Panstruga R (2017) mlo-based powdery mildew resistance in hexaploid bread. wheat generated by a non-transgenic TILLING approach. Plant Biotechnol J 15:367–378

Bettgenhaeuser J, Hernández-Pinzón I, Dawson AM, Gardiner M, Green P, Taylor J, Smoker M, Ferguson JN, Emmrich P, Hubbard A, Bayles R, Waugh R, Steffenson BJ, Wulff BBH, Dreiseitl A, Ward ER, Moscou MJ (2021) The barley immune receptor Mla recognizes multiple pathogens and contributes to host range dynamics. Nat Commun 12:6915

Bhat RA, Miklis M, Schmelzer E, Schulze-Lefert P, Panstruga R (2005) Recruitment and interaction dynamics of plant penetration resistance components in a plasma membrane microdomain. Proc Natl Acad Sci 102:3135–3140

Büschges R, Hollricher K, Panstruga R, Simons G, Wolter M, Frijters A, van Daelen R, van der Lee T, Diergaarde P, Groenendijk J, Töpsch S, Vos P, Salamini F, Schulze-Lefert P (1997) The barley Mlo gene: a novel control element of plant pathogen resistance. Cell 88:695–705

Dean R, Van Kan JAL, Pretorius ZA, Hammond-Kosack KE, Di Pietro A, Spanu PD, Rudd JJ, Dickman M, Kahmann R, Ellis J, Foster GD (2012) The top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol 12:414–430

Devoto A, Piffanelli P, Nilsson I, Wallin E, Panstruga R, von Heijne G, Schulze-Lefert P (1999) Topology, subcellular localization, and sequence diversity of the Mlo family in plants. J Biol Chem 274:34993–35004

Dreiseitl A (2003) Adaptation of Blumeria graminis f. sp. hordei to barley resistance genes in the Czech Republic in 1971–2000. Plant Soil Environ 49:241–248

Dreiseitl A, Jørgensen JH (2000) Powdery mildew resistance in Czech and Slovak barley cultivars. Plant Breed 119:203–209

Dreiseitl A, Zavřelová M (2018) Identification of barley powdery mildew resistances in gene bank accessions and the use of gene diversity for verifying seed purity and authenticity. PLoS One 13:e0208719

Gao Q, Wang C, Xi Y, Shao Q, Li L, Luan S (2022) A receptor-channel trio conducts Ca2+ signaling for pollen tube reception. Nature 607:534–539

Gil-Humanes J, Wang Y, Liang Z, Shan Q, Ozuna CV, Sánchez-León S, Baltes NJ, Starker C, Barro F, Gao C, Voytas DF (2017) High-efficiency gene targeting in hexaploid wheat using DNA replicons and CRISPR/Cas9. Plant J 89:1251–1262

Gu M, Huang H, Hisano H, Ding G, Huang S, Mitani-Ueno N, Yokosho K, Sato K, Yamaji N, Ma JF (2022) A crucial role for a node-localized transporter, HvSPDT, in loading phosphorus into barley grains. New Phytol 234:1249–1261

Hisano H, Sato K (2016) Genomic regions responsible for amenability to Agrobacterium-mediated transformation in barley. Sci Rep 6:37505

Hisano H, Abe F, Hoffie RE, Kumlehn J (2021) Targeted genome modifications in cereal crops. Breed Sci 71:405–416

Hisano H, Hoffie RE, Abe F, Munemori H, Matsuura T, Endo M, Mikami M, Nakamura S, Kumlehn J, Sato K (2022) Regulation of germination by targeted mutagenesis of grain dormancy genes in barley. Plant Biotechnol J 20:37–46

Hückelhoven R, Fodor J, Trujillo M, Kogel KH (2000) Barley Mla and Rar mutants compromised in the hypersensitive cell death response against Blumeria graminis f. sp. hordei are modified in their ability to accumulate reactive oxygen intermediates at sites of fungal invasion. Planta 212:16–24

Jørgensen JH (1992) Discovery, characterization and exploitation of Mlo powdery mildew resistance in barley. Euphytica 63:141–152

Kusch S, Panstruga R (2017) mlo-Based resistance: an apparently universal “weapon” to defeat powdery mildew disease. Mol Plant Microbe Interact 30:179–189

Li S, Lin D, Zhang Y, Deng M, Chen Y, Lv B, Li B, Lei Y, Wang Y, Zhao L, Liang Y, Liu J, Chen K, Liu Z, Xiao J, Qiu JL, Gao C (2022) Genome-edited powdery mildew resistance in wheat without growth penalties. Nature 602:455–460

Mikami M, Toki S, Endo M (2015) Comparison of CRISPR/Cas9 expression constructs for efficient targeted mutagenesis in rice. Plant Mol Biol 88:561–572

Müller J, Piffanelli P, Devoto A, Miklis M, Elliott C, Ortmann B, Schulze-Lefert P, Panstruga R (2005) Conserved ERAD-like quality control of a plant polytopic membrane protein. Plant Cell 17:149–163

Omasits U, Ahrens CH, Müller S, Wollscheid B (2014) Protter: interactive protein feature visualization and integration with experimental proteomic data. Bioinformatics 30:884–886

Pérez-García A, Romero D, Fernández-Ortuño D, López-Ruiz F, De Vicente A, Torés JA (2009) The powdery mildew fungus Podosphaera fusca (synonym Podosphaera xanthii), a constant threat to cucurbits. Mol Plant Pathol 10:153–160

Piffanelli P, Zhou F, Casais C, Orme J, Jarosch B, Schaffrath U, Collins NC, Panstruga R, Schulze-Lefert P (2002) The barley MLO modulator of defense and cell death is responsive to biotic and abiotic stress stimuli. Plant Physiol 129:1076–1085

Reinstädler A, Müller J, Czembor JH, Piffanelli P, Panstruga R (2010) Novel induced mlo mutant alleles in combination with site-directed mutagenesis reveal functionally important domains in the heptahelical barley Mlo protein. BMC Plant Biol 10:31

Spanu PD (2022) Slicing the cost of bread. Nat Plants 8:200–201

Sugai K, Inoue H, Inoue C, Sato M, Wakazaki M, Kobayashi K, Nishiguchi M, Toyooka K, Yamaoka N, Yaeno T (2020) High humidity causes abnormalities in the process of appressorial formation of Blumeria graminis f. sp. hordei. Pathogens 9:45

Ueda K, Nakajima Y, Inoue H, Kobayashi K, Nishiuchi T, Kimura M, Yaeno T (2021) Nicotinamide mononucleotide potentiates resistance to biotrophic invasion of fungal pathogens in barley. Int J Mol Sci 22:2696

Wahara M, Inoue C, Kohguchi T, Sugai K, Kobayashi K, Nishiguchi M, Yamaoka N, Yaeno T (2017) Improved method for in situ biolistic transformation to analyze barley–powdery mildew interactions. J Gen Plant Pathol 83:140–146

Wang Y, Cheng X, Shan Q, Zhang Y, Liu J, Gao C, Qiu JL (2014) Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol 32:947–951

Wei F, Wing RA, Wise RP (2002) Genome dynamics evolution of the Mla (powdery mildew) resistance locus in barley. Plant Cell 14:1903–1917

Wong N, Liu W, Wang X (2015) WU-CRISPR: characteristics of functional guide RNAs for the CRISPR/Cas9 system. Genome Biol 16:218

Yaeno T, Iba K (2008) BAH1/NLA, a RING-type ubiquitin E3 ligase, regulates the accumulation of salicylic acid and immune responses to Pseudomonas syringae DC3000. Plant Physiol 148:1032–1041

Yaeno T, Wahara M, Nagano M, Wanezaki H, Toda H, Inoue H, Eishima A, Nishiguchi M, Hisano H, Kobayashi K, Sato K, Yamaoka N (2021) RACE1, a Japanese Blumeria graminisf. sp.hordei isolate, is capable of overcoming partially mlo-mediated penetration resistance in barley in an allele-specific manner. PLoS One 16:e0256574

Thông tin
Thông tin xuất bản

Springer Science and Business Media LLC

Thông tin tác giả